Pgem T Protocol

TECHNICAL MANUAL
pGEM®-T and pGEM®-T

Easy Vector Systems
Instructions for Use of Products
A1360, A1380, A3600 and A3610
Revised 6/15
TM042
pGEM®-T and pGEM®-T Easy
Vector Systems
All technical literature is available at: www.promega.com/protocols/
Visit the web site to verify that you are using the most current version of this Technical Manual.
E-mail Promega Technical Services if you have questions on use of this system: techserv@promega.com
1. Introduction........................................................................................................................................ 2
1.A. Vector Features........................................................................................................................... 2
1.B. Important Considerations for Successful T-Vector Cloning.............................................................. 2
2. Product Components and Storage Conditions......................................................................................... 3
3. Protocol for Ligations Using the pGEM®-T and pGEM®-T Easy Vectors and the 2X Rapid Ligation Buffer... 4
3.A. Ligation Protocol......................................................................................................................... 4
3.B. Optimizing Insert:Vector Molar Ratios.......................................................................................... 5
4. Transformations Using the pGEM®-T and pGEM®-T Easy Vector Ligation Reactions................................. 6
4.A. Transformation Protocol.............................................................................................................. 6
4.B. Example of Transformation Efficiency Calculation.......................................................................... 7
4.C. Screening Transformants for Inserts............................................................................................. 8
5. pGEM®-T and pGEM®-T Easy Vector Sequences, Multi-Cloning Sites and Circle Maps.............................. 8
5.A. Sequence and Multi-Cloning Site of the pGEM®-T Vector................................................................ 8
5.B. pGEM®-T Vector Map and Sequence Reference Points.................................................................... 9
5.C. Sequence and Multi-Cloning Site of the pGEM®-T Easy Vector...................................................... 10
5.D. pGEM®-T Easy Vector Map and Sequence Reference Points.......................................................... 11
6 General Considerations for PCR Cloning.............................................................................................. 12

6.A. PCR Product Purity.................................................................................................................... 12
6.B. Properties of Various Thermostable Polymerases......................................................................... 12
6.C. Cloning Blunt-Ended PCR Products............................................................................................ 13
7. Experimental Controls........................................................................................................................ 15
8. Troubleshooting................................................................................................................................ 16
9. References......................................................................................................................................... 20
10. Appendix........................................................................................................................................... 20
10.A. pGEM®-T Vector Restriction Enzyme Sites.................................................................................. 20
10.B. pGEM®-T Easy Vector Restriction Enzyme Sites........................................................................... 22
10.C. Composition of Buffers and Solutions.......................................................................................... 24
10.D.Related Products....................................................................................................................... 25
10.E. Summary of Changes................................................................................................................. 27
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516 1
www.promega.com TM042 · Revised 6/15
1. Introduction
1.A. Vector Features

T-Overhangs for Easy PCR Cloning: The pGEM®-T and pGEM®-T Easy Vectors are linearized vectors with a single
3´-terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the efficiency of ligation of
PCR products by preventing recircularization of the vector and providing a compatible overhang for PCR products
generated by certain thermostable polymerases (1,2).
Blue/White Selection of Recombinants: The pGEM®-T and pGEM®-T Easy Vectors are high-copy-number vectors
containing T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding
region of the enzyme β-galactosidase. Insertional inactivation of the α-peptide allows identification of recombinants by
blue/white screening on indicator plates.
Choice of Restriction Sites for Release of Insert: Both the pGEM®-T and pGEM®-T Easy Vectors contain
numerous restriction sites within the multiple cloning region. The pGEM®-T Easy Vector multiple cloning region is
flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing three single-enzyme
digestions for release of the insert. The pGEM®-T Vector cloning region is flanked by recognition sites for the enzyme
BstZI. Alternatively, a double-digestion may be used to release the insert from either vector.
Rapid Ligation: The pGEM®-T and pGEM®-T Easy Vector Systems are supplied with 2X Rapid Ligation Buffer.
Ligation reactions using this buffer may be incubated for 1 hour at room temperature. The incubation period may be
extended to increase the number of colonies after transformation. Generally, an overnight incubation at 4°C produces
the maximum number of transformants.
1.B. Important Considerations for Successful T-Vector Cloning

Avoid introduction of nucleases, which may degrade the T-overhangs on the vector. Use only the T4 DNA Ligase
provided with the system, as this has been tested for minimal exonuclease activity. Use sterile, nuclease-free water in
your ligation reactions.
Use high-efficiency competent cells (≥1 × 108cfu/µg DNA) for transformations. The ligation of fragments
with a single-base overhang can be inefficient, so it is essential to use cells with a transformation efficiency of at least
1 × 108cfu/µg DNA in order to obtain a reasonable number of colonies. However, use of super high-efficiency
competent cells (e.g., XL10 Gold® Cells) may result in a higher background of blue colonies.
Limit exposure of your PCR product to shortwave UV light to avoid formation of pyrimidine dimers. Use a glass
plate between the gel and UV source. If possible, only visualize the PCR product with a long-wave UV source.
2 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM042 · Revised 6/15 www.promega.com
2. Product Components and Storage Conditions
PRODUCT SIZE C A T. #
pGEM®-T Vector System I 20 reactions A3600

Includes:
• 1.2µg pGEM®-T Vector (50ng/µl)
• 12µl Control Insert DNA (4ng/µl)
• 100u T4 DNA Ligase
• 200µl 2X Rapid Ligation Buffer, T4 DNA Ligase
pGEM®-T Vector System II 20 reactions A3610

Includes:
• 1.2µg pGEM®-T Vector (50ng/µl)
• 1.2ml JM109 Competent Cells, High Efficiency (6 × 200µl)
pGEM®-T Easy Vector System I 20 reactions A1360

Includes:
• 1.2µg pGEM®-T Easy Vector (50ng/µl)
pGEM®-T Easy Vector System II 20 reactions A1380

Includes:
• 1.2µg pGEM®-T Easy Vector (50ng/µl)
• 1.2ml JM109 Competent Cells, High Efficiency (6 × 200µl)
Storage Conditions: For Cat.# A3610, A1380, store the Competent Cells at –70°C. Store all other components at
–20°C.
3. Protocol for Ligations Using the pGEM®-T and pGEM®-T Easy Vectors and the 2X Rapid Ligation
Buffer
3.A. Ligation Protocol

1. Briefly centrifuge the pGEM®-T or pGEM®-T Easy Vector and Control Insert DNA tubes to collect the contents
at the bottom of the tubes.
2. Set up ligation reactions as described below.
Note: Use 0.5ml tubes known to have low DNA-binding capacity (e.g., VWR Cat.# 20170-310).
Vortex the 2X Rapid Ligation Buffer vigorously before each use.
3. Mix the reactions by pipetting. Incubate the reactions for 1 hour at room temperature.
Alternatively, if the maximum number of transformants is required, incubate the reactions overnight at 4°C.
Standard Positive Background

Reaction Component Reaction Control Control
2X Rapid Ligation Buffer, T4 DNA Ligase 5µl 5µl 5µl
pGEM -T or pGEM -T Easy Vector (50ng)
® ®
1µl 1µl 1µl
PCR product Xµl* – –
Control Insert DNA – 2µl –
T4 DNA Ligase (3 Weiss units/µl) 1µl 1µl 1µl
nuclease-free water to a final volume of 10µl 10µl 10µl
*Molar ratio of PCR product:vector may require optimization.
Notes:
1. Use only the T4 DNA Ligase supplied with this system to perform pGEM®-T and pGEM®-T Easy Vector
ligations. Other commercial preparations of T4 DNA ligase may contain exonuclease activities that may remove
the terminal deoxythymidines from the vector.
2. 2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-
thaw cycles and exposure to frequent temperature changes by making single-use aliquots of the buffer.
3. Longer incubation times will increase the number of transformants. Generally, incubation overnight at 4°C will
produce the maximum number of transformants.
4. An aliquot of the PCR reaction should be analyzed on an agarose gel before use in the ligation reaction to verify
that the reaction produced the desired product. The PCR product to be ligated can be gel-purified or purified
directly from the PCR amplification using the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281). Clean-
up of reactions prior to ligation is recommended to remove primer dimers or other undesired reaction products,
and to improve ligation efficiency. Exposure of PCR products to shortwave ultraviolet light should be minimized
in order to avoid the formation of pyrimidine dimers.
3.B. Optimizing Insert:Vector Molar Ratios

The pGEM®-T and pGEM®-T Easy Vector Systems have been optimized using a 1:1 molar ratio of the Control Insert
DNA to the vectors. However, ratios of 8:1 to 1:8 have been used successfully. If initial experiments with your PCR
product are suboptimal, ratio optimization may be necessary. Ratios from 3:1 to 1:3 provide good initial parameters.
The concentration of PCR product should be estimated by comparison to DNA mass standards on a gel or by using a
fluorescent assay (3). The pGEM®-T and pGEM®-T Easy Vectors are approximately 3kb and are supplied at 50ng/µl.
To calculate the appropriate amount of PCR product (insert) to include in the ligation reaction, use the following
equation.
ng of vector × kb size of insert

× insert:vector molar ratio = ng of insert
kb size of vector
Example of insert:vector ratio calculation:
How much 0.5kb PCR product should be added to a ligation in which 50ng of 3.0kb vector will be used if a 3:1
insert:vector molar ratio is desired?
50ng vector × 0.5kb insert 3

× = 25ng insert
3.0kb vector 1
Using the same parameters for a 1:1 insert:vector molar ratio, 8.3ng of a 0.5kb insert would be required.
Tip: The Biomath calculator (www.promega.com/biomath/) can be used to determine the amount of insert
DNA needed. The pGEM®-T Vector size is 3000bp and the pGEM®-T Easy Vector size is 3015bp.
4. Transformations Using the pGEM®-T and pGEM®-T Easy Vector Ligation Reactions
Use high-efficiency competent cells (≥1 × 108cfu/µg DNA) for transformations. Ligation of fragments with a single-
base overhang can be inefficient, so it is essential to use cells with a transformation efficiency of 1 × 108cfu/µg DNA (or
higher) in order to obtain a reasonable number of colonies. We recommend using JM109 High Efficiency Competent
Cells (Cat.# L2001); these cells are provided with the pGEM®-T and pGEM®-T Easy Vector Systems II. Other host
strains may be used, but they should be compatible with blue/white color screening and standard ampicillin selection.
Note: Use of super high-efficiency competent cells (e.g., XL10 Gold® Ultracompetent Cells) may result in a higher
background of blue colonies.
If you are using competent cells other than JM109 High Efficiency Competent Cells purchased from Promega, it is
important that the appropriate transformation protocol be followed. Selection for transformants should be on
LB/ampicillin/IPTG/X-Gal plates (See recipe in Section10.C). For best results, do not use plates that are more than
1 month old.
The genotype of JM109 is recA1, endA1, gyrA96, thi, hsdR17 (rK–,mK+), relA1, supE44, ∆(lac-proAB), [F´, traD36,
proAB, lacIqZ∆M15] (4).
4.A. Transformation Protocol
Materials to Be Supplied by the User

(Solution Compositions are provided in Section 10.C.)
• LB plates with ampicillin/IPTG/X-Gal
• SOC medium
1. Prepare two LB/ampicillin/IPTG/X-Gal plates for each ligation reaction, plus two plates for determining
transformation efficiency. Equilibrate the plates to room temperature.
2. Centrifuge the tubes containing the ligation reactions to collect the contents at the bottom. Add 2µl of each
ligation reaction to a sterile (17 × 100mm) polypropylene tube or a 1.5ml microcentrifuge tube on ice (see
Note 1). Set up another tube on ice with 0.1ng uncut plasmid for determination of the transformation efficiency
of the competent cells.
3. Remove tube(s) of frozen JM109 High Efficiency Competent Cells from storage and place in an ice bath until just
thawed (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent
cells are extremely fragile.
4. Carefully transfer 50µl of cells into each tube prepared in Step 2 (use 100µl of cells for determination of
transformation efficiency).
5. Gently flick the tubes to mix and place them on ice for 20 minutes.
6. Heat-shock the cells for 45–50 seconds in a water bath at exactly 42°C (do not shake).
7. Immediately return the tubes to ice for 2 minutes.
8. Add 950µl room-temperature SOC medium to the tubes containing cells transformed with ligation reactions
and 900µl to the tube containing cells transformed with uncut plasmid (LB broth may be substituted, but colony
number may be lower).
9. Incubate for 1.5 hours at 37°C with shaking (~150rpm).
10. Plate 100µl of each transformation culture onto duplicate LB/ampicillin/IPTG/X-Gal plates. For the transfor-
mation control, a 1:10 dilution with SOC medium is recommended for plating. If a higher number of colonies
is desired, the cells may be pelleted by centrifugation at 1,000 × g for 10 minutes, resuspended in 200µl of
SOC medium, and 100µl plated on each of two plates.
11. Incubate the plates overnight (16–24 hours) at 37°C. If 100µl is plated, approximately 100 colonies per plate are
routinely seen using competent cells that are 1 × 108cfu/µg DNA. Use of ultra-high- efficiency competent cells
may result in a higher number of background colonies. Longer incubations or storage of plates at 4°C (after 37°C
overnight incubation) may be used to facilitate blue color development. White colonies generally contain inserts;
however, inserts may also be present in blue colonies.
Notes:
1. We have found that use of larger (17 × 100mm) polypropylene tubes (e.g., Falcon™ Cat.# 2059) increases
transformation efficiency. Tubes from some manufacturers bind DNA and should be avoided.
2. Colonies containing β-galactosidase activity may grow poorly relative to cells lacking this activity. After overnight
growth, the blue colonies may be smaller than the white colonies, which are approximately one millimeter in
diameter.
3. Blue color will become darker after the plate has been stored overnight at 4ºC.
4.B. Example of Transformation Efficiency Calculation

After 100µl of competent cells are transformed with 0.1ng of uncut plasmid DNA, the transformation reaction is added
to 900µl of SOC medium (0.1ng DNA/ml). From that volume, a 1:10 dilution with SOC medium (0.01ng DNA/ml) is
made and 100µl plated on two plates (0.001ng DNA/100µl). If 200 colonies are obtained (average of two plates), what
is the transformation efficiency?
200cfu
= 2 × 105cfu/ng = 2 × 108cfu/µg DNA
0.001ng
4.C. Screening Transformants for Inserts
Successful cloning of an insert into the pGEM®-T or pGEM®-T Easy Vector interrupts the coding sequence of
β-galactosidase; recombinant clones can be identified by color screening on indicator plates. However, the charac-
teristics of the PCR products cloned into the vectors can significantly affect the ratio of blue:white colonies obtained.
Usually clones containing PCR products produce white colonies, but blue colonies can result from PCR fragments that
are cloned in-frame with the lacZ gene. Such fragments are usually a multiple of 3 base pairs long (including the 3´-A
overhangs) and do not contain in-frame stop codons. There have been reports of DNA fragments up to 2kb that have
been cloned in-frame and have produced blue colonies. Even if your PCR product is not a multiple of 3 bases long, the
amplification process can introduce mutations (deletions or point mutations) that may result in blue colonies.
The Control Insert DNA supplied with the pGEM®-T and pGEM®-T Easy Systems is a 542bp fragment from
pGEM®-luc Vector DNA (Cat.# E1541). This sequence has been mutated to contain multiple stop codons in all six
reading frames, which ensures a low background of blue colonies for the control reaction. Results obtained with the
Control Insert DNA may not be representative of those achieved with your PCR product.
5. pGEM®-T and pGEM®-T Easy Vector Sequences, Multi-Cloning Sites and Circle Maps
5.A. Sequence and Multi-Cloning Site of the pGEM®-T Vector

The pGEM®-T Vector is derived from the pGEM®-5Zf(+) Vector (GenBank® Accession No. X65308). The pGEM®-T
Vector was created by linearizing the pGEM®-5Zf(+) Vector with EcoRV at base 51 and adding a T to both 3´-ends.
The EcoRV site will not be recovered upon ligation of the vector and insert.
T7 Transcription Start
5′ . . . TGTAA TACGA CTCAC TATAG GGCGA ATTGG GCCCG ACGTC GCATG CTCCC GGCCG
3′ . . . ACATT ATGCT GAGTG ATATC CCGCT TAACC CGGGC TGCAG CGTAC GAGGG CCGGC
T7 Promoter
ApaI AatII SphI BstZI
CCATG GCCGC GGGATT3′

GGTAC CGGCG CCCTA (
cloned insert )
ATCAC TAGTG CGGCC GCCTG CAGGT CGACC ATATG
3′TTAGTG ATCAC GCCGG CGGAC GTCCA GCTGG TATAC
NcoI SacII SpeI NotI PstI SalI NdeI

BstZI
SP6 Transcription Start
GGAGA GCTCC CAACG CGTTG GATGC ATAGC TTGAG TATTC TATAG TGTCA CCTAA AT . . . 3′
0357MA06_2A
CCTCT CGAGG GTTGC GCAAC CTACG TATCG AACTC ATAAG ATATC ACAGT GGATT TA . . . 5′
SP6 Promoter
SacI BstXI NsiI
Figure 1. The promoter and multiple cloning sequence of the pGEM®-T Vector. The top strand corresponds
to the RNA synthesized by T7 RNA polymerase. The bottom strand corresponds to the RNA synthesized by SP6 RNA
polymerase.
5.B. pGEM®-T Vector Map and Sequence Reference Points
XmnI 1994
ScaI NaeI
1875 2692
T7
➞
f1 ori 1 start
ApaI 14
AatII 20
SphI 26
Amp r BstZI 31
pGEM®-T lac Z NcoI 37
Vector T T SacII 46
(3000bp)
SpeI 55
NotI 62
BstZI 62
PstI 73
SalI 75
NdeI 82
SacI 94
ori BstXI 103
0356VA04_3A
NsiI 112
126
➞
SP6
pGEM®-T Vector sequence reference points:

T7 RNA polymerase transcription initiation site 1
multiple cloning region 10–113
SP6 RNA polymerase promoter (–17 to +3) 124–143
SP6 RNA polymerase transcription initiation site 126
pUC/M13 Reverse Sequencing Primer binding site 161–177
lacZ start codon 165
lac operator 185–201
β-lactamase coding region 1322–2182
phage f1 region 2365–2820
lac operon sequences 2821–2981, 151–380
pUC/M13 Forward Sequencing Primer binding site 2941–2957
T7 RNA polymerase promoter (–17 to +3) 2984–3
Note: Inserts can be sequenced using the SP6 Promoter Primer (Cat.# Q5011), T7 Promoter Primer (Cat.# Q5021),
pUC/M13 Forward Primer (Cat.# Q5601), or pUC/M13 Reverse Primer (Cat.# Q5421).
Note: A single digest with BstZI will release inserts cloned into the pGEM®-T Vector. Double digests can also be
! used to release inserts. Isoschizomers of BstZI include EagI and Eco52I.
5.C. Sequence and Multi-Cloning Site of the pGEM®-T Easy Vector
The sequence of the pGEM®-T Easy Vector is available at: www.promega.com/vectors/
The pGEM®-T Easy Vector has been linearized at base 60 with EcoRV and a T added to both 3´-ends. The EcoRV site
will not be recovered upon ligation of the vector and insert.
T7 Transcription Start
5′ . . . TGTAA TACGA CTCAC TATAG GGCGA ATTGG GCCCG ACGTC GCATG CTCCC GGCCG CCATG
3′ . . . ACATT ATGCT GAGTG ATATC CCGCT TAACC CGGGC TGCAG CGTAC GAGGG CCGGC GGTAC
T7 Promoter
ApaI AatII SphI BstZI NcoI
GCGGC CGCGG GAATT CGATT3′

CGCCG GCGCC CTTAA GCTA (
cloned insert )
ATCAC TAGTG AATTC GCGGC CGCCT GCAGG TCGAC
3′TTAGTG ATCAC TTAAG CGCCG GCGGA CGTCC AGCTG
NotI NotI
SacII EcoRI SpeI EcoRI PstI SalI
BstZI BstZI
SP6 Transcription Start
CATAT GGGA GAGCT CCCAA CGCGT TGGAT GCATA GCT TG AGTAT TCTAT AGTGT CACCT AAAT . . . 3′
GTATA CCCT CTCGA GGGTT GCGCA ACCTA CGTAT CGAAC T CATA AGATA TCACA GTGGA TT TA . . . 5′
SP6 Promoter
NdeI SacI BstXI NsiI
Figure 2. The promoter and multiple cloning sequence of the pGEM®-T Easy Vector. The top strand shown
corresponds to the RNA synthesized by T7 RNA polymerase. The bottom strand corresponds to the RNA synthesized
by SP6 RNA polymerase.
More PCR Cloning Resources are available in the Cloning Chapter of the Protocols and Applications Guide at:
www.promega.com/paguide/
5.D. pGEM®-T Easy Vector Map and Sequence Reference Points
XmnI 2009
➞
T7 1 start
ScaI 1890 NaeI 2707 ApaI 14
AatII 20
f1 ori SphI 26
BstZI 31
NcoI 37
BstZI 43
Ampr NotI 43
pGEM-T Easy lacZ SacII 49
T
Vector T EcoRI 52
(3015bp)
SpeI 64
EcoRI 70
NotI 77
BstZI 77
PstI 88
ori SalI 90
NdeI 97
SacI 109
BstXI 118
1473VA05_6A
NsiI 127
141
➞
SP6
pGEM®-T Easy Vector sequence reference points:
T7 RNA polymerase transcription initiation site 1

multiple cloning region 10–128
SP6 RNA polymerase promoter (–17 to +3) 139–158
SP6 RNA polymerase transcription initiation site 141
pUC/M13 Reverse Sequencing Primer binding site 176–197
lacZ start codon 180
lac operator 200–216
β-lactamase coding region 1337–2197
phage f1 region 2380–2835
lac operon sequences 2836–2996, 166–395
pUC/M13 Forward Sequencing Primer binding site 2949–2972
T7 RNA polymerase promoter (–17 to +3) 2999–3
Note: Inserts can be sequenced using the SP6 Promoter Primer (Cat.# Q5011), T7 Promoter Primer (Cat.# Q5021),
pUC/M13 Forward Primer (Cat.# Q5601), or pUC/M13 Reverse Primer (Cat.# Q5421).
Note: A single digest with BstZI, EcoRI (Cat.# R6011) or NotI (Cat.# R6431) will release inserts cloned into the
! pGEM®-T Easy Vector. Double digests can also be used to release inserts. Isoschizomers of BstZI include EagI
and Eco52I.
6 General Considerations for PCR Cloning
6.A. PCR Product Purity

An aliquot of the PCR reaction should be analyzed on an agarose gel before use in the ligation reaction. The PCR
product can be gel-purified or purified directly from the PCR amplification using the Wizard® SV Gel and PCR
Clean-Up System (Cat.# A9281). Exposure to shortwave ultraviolet light should be minimized to avoid the formation
of pyrimidine dimers. Even if distinct bands of the expected size are observed, primer-dimers should be removed by
gel purification or by using the Wizard® SV Gel and PCR Clean-Up System. Use of crude PCR product may produce
successful ligation in some cases; however, the number of white colonies containing the relevant insert may be reduced
due to preferential incorporation of primer-dimers or other extraneous reaction products. Therefore, it may be
necessary to screen numerous colonies in order to identify clones that contain the PCR product of interest.
6.B. Properties of Various Thermostable Polymerases

Not all thermostable polymerases generate fragments with 3Á-tailed fragments. Table 1 lists the properties of several
commonly used polymerase enzymes.
Table 1. Comparison of PCR Product Properties for Thermostable DNA Polymerases.
Thermostable DNA Polymerase

GoTaq / ®
Vent® Deep
Taq/ Tfl Tth Pfu Pwo
(Tli) Vent®
Characteristic AmpliTaq®
Resulting DNA ends 3Á 3Á 3Á Blunt Blunt Blunt Blunt
5´→ 3éxonuclease activity Yes Yes Yes No No No No
3´→ 5éxonuclease activity No No No Yes Yes Yes Yes
6.C. Cloning Blunt-Ended PCR Products
Thermostable DNA polymerases with proofreading activity, such as Pfu DNA Polymerase (Cat.# M7741),
Pwo DNA polymerase and Tli DNA Polymerase, generate blunt-ended fragments. Nevertheless, PCR products
generated using these polymerases can be modified using the A-tailing procedure outlined in Figure 3 and ligated into
the pGEM®-T and pGEM®-T Easy Vectors (5). Using this method, only one insert will be ligated into the vector (as
opposed to multiple insertions that can occur with blunt-ended cloning). In addition, with T-vector cloning there is no
need to dephosphorylate the vector, and there is a low background of religated vector.
Using this procedure with optimized insert:vector ratios, 55–95% recombinants were obtained when Pfu and Tli DNA
Polymerases were used to generate the insert DNA (Table 2). It is critical that the PCR fragments are purified using the
Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281) or by direct isolation from a gel by other means. In the
absence of purification, the proofreading activity of the Pfu, Pwo and Tli DNA Polymerases will degrade the PCR
fragments, or remove the 3´-terminal deoxyadenosine added during tailing or the 3´-terminal deoxythymidine from
the vector during the A-tailing reaction or ligation.
To optimize cloning efficiency, the amount of DNA in the A-tailing reaction and the ligation volumes must be adjusted
depending on the molar yield of the purified PCR product. When molar concentrations are high due to small fragment
size and/or good amplification, small volumes of the PCR fragment are needed for the A-tailing and ligation reactions.
However, when molar concentration is low due to large fragment size and/or poor amplification, large volumes of the
PCR fragment are needed for the A-tailing and ligation reactions. We have successfully used 1–7µl of purified PCR
fragment in A-tailing reactions to optimize the insert:vector ratio. (See Section 3.B for further discussion of optimizing
the insert:vector ratio.) Recombinants were identified by blue/white screening, and 70–100% were shown to have the
correct size insert by PCR. Few recombinants were observed in control reactions in which the PCR fragment was not
tailed. These control results confirm that the majority of the pGEM®-T Easy Vector used contained 3´-terminal
deoxythymidine and that, during the A-tailing, Taq DNA Polymerase added a 3´-terminal deoxyadenosine to a
significant proportion of the PCR fragments.
6.C. Cloning Blunt-Ended PCR Products (continued)
Table 2. Comparison of A-Tailing Procedures.
% Recombinants1
1-Hour Ligation at 24°C 16-Hour Ligation at 4°C
(Standard) (Alternative)
Polymerase 542bp 1.8kb 542bp 1.8kb
Pfu DNA Polymerase 65–84%2 31–55%3 81–95%2 50–75%3
Tli DNA Polymerase 68–77%4 37–65%5 85–93%4 60–81%5
PCR fragments generated by Pfu and Tli DNA Polymerase were A-tailed and ligated into pGEM®-T Easy Vector for
1 hour at 24°C or for 16 hours at 4°C. Two microliters of ligation mix was transformed into JM109 Competent Cells
and plated on LB/amp/IPTG/X-gal plates.
% Recombinants = % white and/or pale blue colonies. PCR fragments were purified with the Wizard® PCR Preps
1
DNA Purification System prior to A-tailing.

2
Insert:vector ratios tested: 5:1, 3:1, 1:1. Volume of PCR amplification product used in A-tailing: 1–2µl.
3
4
5
Insert:vector ratios tested: 2:1, 1:1. Volume of PCR amplification product used in A-tailing: 4–7µl.
Start with 1–7µl of purified PCR fragment generated by a

proofreading polymerase (e.g., Pfu DNA Polymerase).
Add 1µl Taq DNA Polymerase 10X Reaction Buffer with MgCl2.
Add dATP to a final concentration of 0.2mM.
Add 5 units of Taq DNA Polymerase.
Add deionized water to a final reaction volume of 10µl.
Incubate at 70°C for 15–30 minutes.

2357MA02_9A
Use 1–2µl in a ligation reaction with

Promega’s pGEM®-T and pGEM®-T Easy Vector.
Figure 3. An A-tailing procedure for blunt-ended PCR fragments purified with the Wizard® SV Gel and
PCR Clean-Up System (Cat.# A9281) and used in T-vector cloning.
7. Experimental Controls
Positive Control: Set up a ligation reaction with the Control Insert DNA as described in Section 3 and use it for
transformations. This control will allow you to determine whether the ligation is proceeding efficiently. Typically,
approximately 100 colonies should be observed, 10–40% of which are blue, when competent cells that have a
transformation efficiency of 1 × 108cfu/µg DNA are transformed. Greater than 60% of the colonies should be white.
The Control Insert DNA is designed to produce white colonies; however, other insert DNA may not yield white colonies
(see Section 4.C). Background blue colonies from the positive control ligation reaction arise from non-T-tailed or
undigested pGEM®-T or pGEM®-T Easy Vector. These blue colonies are a useful internal transformation control; if no
colonies are obtained, the transformation has failed. If small numbers of blue colonies are obtained, but no whites, the
ligation reaction may have failed. If <50% white colonies are seen in the positive control reaction, then the ligation
conditions were probably suboptimal or nuclease contamination of the ligation reaction may have occurred.
The concentration of the Control Insert DNA is such that 2µl (4ng/µl) can be used in a 10µl ligation reaction to achieve
a 1:1 molar ratio with 50ng of the pGEM®-T or pGEM®-T Easy Vectors.
Background Control: Set up a ligation reaction with 50ng of pGEM®-T or pGEM®-T Easy Vector and no insert as
described in Section 3, and use it for transformations. This control allows determination of the number of background
blue colonies resulting from non-T-tailed or undigested pGEM®-T or pGEM®-T Easy Vector alone. If the recom-
mendations in Section 4 are followed, 10–30 blue colonies will typically be observed if the transformation efficiency of
the competent cells is 1 × 108cfu/µg DNA. (Under these conditions, cells that have an efficiency of 1 × 107cfu/µg DNA
would yield 1–3 blue colonies, and cells with a transformation efficiency of 1 × 109cfu/µg DNA would yield 100–300
blue colonies). Compare the number of blue colonies obtained with this background control to the number of blue
colonies obtained in the standard reaction using the PCR product. If ligation of the PCR product yields dramatically
more blue colonies than the background control reaction, then recombinants are probably among these blue colonies
(see Section 4.C).
Transformation Control: Check the transformation efficiency of the competent cells by transforming them with an
uncut plasmid (not pGEM®-T or pGEM®-T Easy, since these vectors are linearized) and calculating cfu/µg DNA. If the
transformation efficiency is lower than 1 × 108cfu/µg DNA, prepare fresh cells. If you are not using JM109 High
Efficiency Competent Cells (provided with pGEM®-T and pGEM®-T Easy Vector Systems II; Cat.# A3610 and A1380,
respectively), be sure the cells are compatible with blue/white screening and standard ampicillin selection and have a
transformation efficiency of at least 1 × 108cfu/µg DNA.
8. Troubleshooting
For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information
available at: www.promega.com. E-mail: techserv@promega.com
Symptoms Causes and Comments

No colonies A problem has occurred with the transformation reaction or the
cells have lost competence. Background undigested vector and
religated non-T-tailed vector should yield 10–30 blue colonies
independent of the presence of insert DNA. Check the back-
ground control (Section 7).
Use high-efficiency competent cells (≥1 × 108cfu/µg DNA). Test
the efficiency by transforming the cells with an uncut plasmid
that allows for antibiotic selection, such as the pGEM®-5Zf(+)
Vector. If the guidelines in Section 4 are followed, cells at
1 × 108cfu/µg DNA typically yield 100 colonies. Therefore, you
would not see any colonies from cells that are <1 × 107cfu/µg
DNA (Section 7).
Less than 10% white colonies Improper dilution of the 2X Rapid Ligation. The T4 DNA ligase
with Control Insert DNA buffer is provided at a concentration of 2X. Use 5µl in a 10µl
reaction.
If the total number of colonies is high, but there are few/no
white colonies, competent cells may be high efficiency
(≥1 × 109cfu/µg), but there may be a ligation problem.
Approximately 1,000 colonies can be obtained from the
positive control ligation using cells that are 109cfu/µg DNA,
with 70–90% white colonies. If ligation is suboptimal or fails,
the total number of colonies will be high (up to 300 cells at
1 × 109cfu/µg), but the amount of white colonies will be low or
zero.
Less than 10% white colonies with Ligation reaction has failed. Ligase buffer may DNA have low
Control Insert DNA (continued) activity. The 2X Rapid Ligation Buffer contains ATP, which
degrades during temperature fluctuations. Avoid multiple
freeze-thaw cycles by making single-use aliquots of the buffer.
Use a fresh vial of buffer. To test the activity of the ligase and
buffer, set up a ligation with ~20ng of DNA markers (e.g.,
Lambda DNA/HindIII Markers, Cat.# G1711). Compare ligated
and nonligated DNA on a gel and check that the fragments have
been religated into high-molecular-weight material.
T-overhangs have been removed, allowing blunt-ended ligation
of vector and giving rise to more blue than white colonies. Avoid
introduction of nucleases, which may degrade the T-overhangs.
Use only the T4 DNA Ligase provided with the system, which
has been tested for minimal exonuclease activity. Also, use
sterile, nuclease-free water.
Less than 60% white colonies with Improper dilution of the Rapid Ligation Buffer. The Rapid
Control Insert DNA Ligation Buffer is provided at a 2X concentration. Use 5µl in a
10µl reaction.
T-overhangs have been removed, allowing blunt-ended ligation
of vector and giving rise to more blue than white colonies. Avoid
introduction of nucleases, which may degrade the T-overhangs.
Use only the T4 DNA Ligase provided with the system, which
has been tested for minimal exonuclease activity.
Ligation temperature is too high. Higher temperatures (>28°C)
give rise to increased background and fewer recombinants.
Low number or no white colonies Improper dilution of the Rapid Ligation Buffer. The Rapid
containing PCR product Ligation Buffer is provided at a 2X concentration. Use 5µl in a
10µl reaction.
Ligation incubation is not long enough. Optimal results are seen
with an overnight ligation.
Failed ligation due to an inhibitory component in the PCR
product. Mix some of the PCR product with the positive control
ligation to determine whether an inhibitor is present. If an
inhibitor is indicated, repurify the PCR fragment.
8. Troubleshooting (continued)

Low number or no white colonies PCR product is not ligating because there are no 3´-A overhangs.
containing PCR product (continued) As summarized in Table 1, not all thermostable DNA polymer-
ases create a 3´-A overhang (6,7). Blunt-ended fragments may
be subsequently A-tailed by treatment with an appropriate
polymerase and dATP (8–10).
PCR product cannot be ligated due to pyrimidine dimers formed
from UV over-exposure. This is a common problem with
gel-purified DNA. There is no way to fix this; the DNA must be
remade. Exposure to shortwave UV should be limited as much as
possible. Use a glass plate between the gel and UV source to
decrease UV overexposure. If possible, only visualize the PCR
product using a longwave UV source.
The PCR fragment is inserted, but it is not disrupting the lacZ
gene. If there are a higher number of blue colonies resulting
from the PCR fragment ligation than with the background
control, some of these blue colonies may contain insert. Screen
blue and pale blue colonies (see Section 4.C).
Insert:vector ratio is not optimal. Check the integrity and
quantity of your PCR fragment by gel analysis. Optimize the
insert:vector ratio (see Section 3.B).
There may be primer-dimers present in PCR fragment prepara-
tion. Primer-dimers will ligate into the pGEM®-T or pGEM®-T
Easy Vector but may not be seen after restriction digestion and
gel analysis because of their small size. The vector will appear to
contain no insert. More blue colonies may be seen with the
ligation than on the background control plates. The PCR
fragment should be gel-purified.
Multiple PCR products may have been generated and cloned
into the pGEM®-T or pGEM®-T Easy Vector. Gel-purify the
PCR fragment of interest.
Low number or no white colonies DNA has rearranged. Check a number of clones to see whether
containing PCR product (continued) the rearrangement is random. If so, the clone of interest should
be present and can be identified by screening several clones. If
the same rearrangement is found in all of the clones, use of a
repair-deficient bacterial strain (e.g., SURE® cells) may reduce
recombination events.
PCR product ligation reaction produces Ampicillin is inactive, allowing ampicillin- sensitive cells to grow.
white colonies only (no blue colonies) Check that ampicillin plates are made properly and used within
one month. Test ampicillin activity by streaking plates, with and
without ampicillin, using an ampicillin-sensitive clone.
The bacterial strain (e.g., JM109) has lost its F´ episome, or
the bacterial strain used is not compatible with blue/white
screening. Check the background control. If these colonies are
not blue, the cells may have lost the F´ episome (assuming
lacIqZ∆M15 is located on the F´ in the transformed strain and
appropriate plates were used). Be sure that the cells are
prepared properly for use with this system (see Section 4).
Plates are incompatible with blue/white screening. Check the
background control. If these colonies are not blue, check that
the plates have ampicillin/IPTG/X-Gal and are fresh. If there is
any question about the quality of the plates, repeat plating with
fresh plates.
Not enough clones contain the Insufficient A-tailing of the PCR fragment. After the PCR
PCR product of interest product of interest urification of the PCR fragment, set up an
A-tailing reaction (8–10). Clean up the sample and proceed with
the protocol.
Insert:vector ratio is not optimal. Check the integrity and quality
of your PCR fragment by gel analysis. Optimize the insert:vector
ratio (see Section 3.B).
Multiple PCR products are generated and cloned into the
pGEM®-T or pGEM®-T Easy Vector. Gel-purify the PCR
fragment of interest.
9. References
1. Mezei, L.M. and Storts, D.R. (1994) Purification of PCR products. In: PCR Technology: Current Innovations,
Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL, 21.
2. Robles, J. and Doers, M. (1994) pGEM®-T Vector Systems troubleshooting guide. Promega Notes 45, 19–20.
3. Haff, L. and Mezei, L. (1989) Amplifications 1, 8.
4. Messing, J. et al. (1981) A system for shotgun DNA sequencing. Nucl. Acids Res. 9, 309–21.
5. Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA Polymerase-generated PCR fragments into
pGEM®-T Vector Systems. Promega Notes 71, 10–13.
6. Clark, J.M. (1988) Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic
DNA polymerases. Nucl. Acids Res. 16, 9677–86.
7. Newton, C.R. and Graham, A. (1994) In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK, 13.
8. Kobs, G. (1995) pGEM®-T Vector: Cloning of modified blunt-ended DNA fragments. Promega Notes 55,
28–29.
9. Kobs, G. (1997) Cloning blunt-end DNA fragments into the pGEM®-T Vector Systems. Promega Notes 62,
15–18.
10. Zhou, M.-Y., Clark, S.E. and Gomez-Sanchez, C.E. (1995) Universal cloning method by TA strategy. BioTech-
niques 19, 34–35.
10. Appendix
10.A. pGEM®-T Vector Restriction Enzyme Sites

The pGEM®-T Vector is derived from the circular pGEM®-5Zf(+) Vector (GenBank® Accession No. X65308). The
pGEM®-5Zf(+) Vector sequence is available at: www.promega.com/vectors/
The following restriction enzyme tables are based on those of the circular pGEM®-5Zf(+) Vector. The pGEM®-T Vector
has been created by linearizing the pGEM®-5Zf(+) Vector with EcoRV at base 51 and adding a T to both 3´-ends. This
site will not be recovered upon ligation of the vector and insert. The following tables were constructed using
DNASTAR® sequence analysis software. Please note that we have not verified this information by restriction digestion
with each enzyme listed. The location given specifies the 3´-end of the cut DNA (the base to the left of the cut site).
Please contact your local Promega Branch Office or Distributor if you identify a discrepancy. In the U.S., contact
Technical Services at 1-800-356-9526.
Table 3. Restriction Enzymes That Cut the pGEM®-T Vector 1–5 Times.
Enzyme # of Sites Location Enzyme # of Sites Location

AatII 1 20 FokI 5 119, 1361, 1542, 1829, 2919
AccI 1 76 FspI 2 1617, 2840
AcyI 2 17, 1932 HaeII 4 380, 750, 2740, 2748
AflIII 2 99, 502 HgaI 4 613, 1191, 1921, 2806
Alw26I 2 1456, 2232 HincII 1 77
Alw44I 2 816, 2062 HindII 1 77
AlwNI 1 918 Hsp92I 2 17, 1932
ApaI 1 14 MaeI 5 56, 997, 1250, 1585, 2740
AspHI 4 94, 820, 1981, 2066 MluI 1 99
AvaII 2 1533, 1755 NaeI 1 2692
BanI 3 246, 1343, 2626 NciI 4 30, 882, 1578, 1929
BanII 3 14, 94, 2664 NcoI 1 37
BbuI 1 26 NdeI 1 82
BglI 3 39, 1515, 2833 NgoMIV 1 2690
BsaI 1 1456 NotI 1 62
BsaAI 1 2589 NsiI 1 112
BsaHI 2 17, 1932 NspI 2 26, 506
BsaJI 5 37, 43, 241, 662, 2936 Ppu10I 1 108
Bsp120I 1 10 PstI 1 73
BspHI 2 1222, 2230 PvuI 2 1765, 2861
BspMI 1 62 PvuII 2 326, 2890
BssSI 2 675, 2059 RsaI 1 1875
BstOI 5 242, 530, 651, 664, 2937 SacI 1 94
BstXI 1 103 SacII 1 46
BstZI 2 31, 62 SalI 1 75
Cfr10I 2 1475, 2690 ScaI 1 1875
DdeI 4 777, 1186, 1352, 1892 SfiI 1 39
DraI 3 1261, 1280, 1972 SinI 2 1533, 1755
DraIII 1 2589 SpeI 1 55
DrdI 2 610, 2544 SphI 1 26
DsaI 2 37, 43 Sse8387I 1 73
EagI 2 31, 62 SspI 2 2199, 2381
EarI 3 386, 2190, 2878 StyI 1 37
EclHKI 1 1395 TaqI 4 76, 602, 2046, 2622
Eco52I 2 31, 62 TfiI 2 337, 477
EcoICRI 1 92 VspI 3 273, 332, 1567
EcoRV 1 51* XmnI 1 1994
*The pGEM®-T Vector has been created by linearizing the pGEM®-5Zf(+) Vector with EcoRV at base 51 and adding a
T to both 3´-ends. This site will not be recovered upon ligation of the vector and insert.
Note: The enzymes listed in boldface type are available from Promega.
10.A.pGEM®-T Vector Restriction Enzyme Sites (continued)
Table 4. Restriction Enzymes That Do Not Cut the pGEM®-T Vector.
AccB7I BbsI BstEII FseI PinAI SplI

AccIII BclI Bsu36I HindIII PmeI SrfI
Acc65I BglII ClaI HpaI PmlI StuI
AflII BlpI CspI I-PpoI PpuMI SwaI
AgeI Bpu1102I Csp45I KasI PshAI Tth111I
AscI BsaBI DraII KpnI Psp5II XbaI
AvaI BsaMI Eco47III NarI PspAI XcmI
AvrII BsmI Eco72I NheI RsrII XhoI
BalI BsrGI Eco81I NruI SgfI XmaI
BamHI BssHII EcoNI PacI SgrAI
BbeI Bst1107I EcoRI PaeR7I SmaI
BbrPI Bst98I EheI PflMI SnaBI
10.B.pGEM®-T Easy Vector Restriction Enzyme Sites
The sequence of the pGEM®-T Easy Vector is available on the Internet at: www.promega.com/vectors/
The pGEM®-T Easy Vector has been linearized at base 60 with EcoRV and a T added to both 3´-ends. This site will not
be recovered upon ligation of the vector and insert. The following tables were constructed using DNASTAR® sequence
analysis software. Please note that we have not verified this information by restriction digestion with each enzyme
listed. The location given specifies the 3´-end of the cut DNA (the base to the left of the cut site). Please contact your
local Promega Branch Office or Distributor if you identify a discrepancy. In the U.S., contact Technical Services at
1-800-356-9526.
Table 5. Restriction Enzymes that Cut the pGEM®-T Easy Vector 1–5 Times.
Enzyme # of Sites Location Enzyme # of Sites Location

AatII 1 20 EcoRV 1 60*
AccI 1 91 FokI 5 134, 1376, 1557, 1844, 2931
AcyI 2 17, 1947 FspI 2 1632, 2855
AflIII 2 114, 517 HaeII 4 395, 765, 2755, 2763
Alw26I 2 1471, 2247 HgaI 4 628, 1206, 1936, 2821
Alw44I 2 831, 2077 HincII 1 92
AlwNI 1 933 HindII 1 92
ApaI 1 14 Hsp92I 2 17, 1947
AspHI 4 109, 835, 1996, 2081 MaeI 5 65, 1012, 1265, 1600, 2755
AvaII 2 1548, 1770 MluI 1 114
BanI 3 261, 1358, 2641 NaeI 1 2707
BanII 3 14, 109, 2679 NciI 4 30, 897, 1593, 1944
BbuI 1 26 NcoI 1 37
BglI 4 39, 42, 1530, 2848 NdeI 1 97
BsaI 1 1471 NgoMIV 1 2705
BsaAI 1 2604 NotI 2 43, 77
BsaHI 2 17, 1947 NsiI 1 127
BsaJI 5 37, 46, 256, 677, 2951 NspI 2 26, 521
Bsp120I 1 10 Ppu10I 1 123
BspHI 2 1237, 2245 PstI 1 88
BspMI 1 77 PvuI 2 1780, 2876
BssSI 2 690, 2074 PvuII 2 341, 2905
BstOI 5 257, 545, 666, 679, 2952 RsaI 1 1890
BstXI 1 118 SacI 1 109
BstZI 3 31, 43, 77 SacII 1 49
Cfr10I 2 1490, 2705 SalI 1 90
DdeI 4 792, 1201, 1367, 1907 ScaI 1 1890
DraI 3 1276, 1295, 1987 SinI 2 1548, 1770
DraIII 1 2604 SpeI 1 64
DrdI 2 625, 2559 SphI 1 26
DsaI 2 37, 46 Sse8387I 1 88
EagI 3 31, 43, 77 SspI 2 2214, 2396
EarI 3 401, 2205, 2893 StyI 1 37
EclHKI 1 1410 TaqI 5 56, 91, 617, 2061, 2637
Eco52I 3 31, 43, 77 TfiI 2 352, 492
EcoICRI 1 107 VspI 3 288, 347, 1582
EcoRI 2 52, 70 XmnI 1 2009
*The pGEM®-T Easy Vector has been linearized at base 60 with EcoRV and a T added to both 3´-ends. This site will
not be recovered upon ligation of the vector and insert.
10.B.pGEM®-T Easy Vector Restriction Enzyme Sites (continued)
Table 6. Restriction Enzymes That Do Not Cut the pGEM®-T Easy Vector.
AccB7I BbsI BstEII HindIII PmeI SplI

AccIII BclI Bsu36I HpaI PmlI SrfI
Acc65I BglII ClaI I-PpoI PpuMI StuI
AflII BlpI CspI KasI PshAI SwaI
AgeI Bpu1102I Csp45I KpnI Psp5II Tth111I
AscI BsaBI DraII NarI PspAI XbaI
AvaI BsaMI Eco47III NheI RsrII XcmI
AvrII BsmI Eco72I NruI SfiI XhoI
BalI BsrGI Eco81I PacI SgfI XmaI
BamHI BssHII EcoNI PaeR7I SgrAI
BbeI Bst1107I EheI PflMI SmaI
BbrPI Bst98I FseI PinAI SnaBI
10.C. Composition of Buffers and Solutions
IPTG stock solution (0.1M) LB plates with ampicillin

1.2g IPTG
Add 15g agar to 1 liter of LB medium. Autoclave. Allow
Add water to 50ml final volume. Filter-sterilize and the medium to cool to 50°C before adding ampicillin to a
store at 4°C. final concentration of 100µg/ml. Pour 30–35ml of
medium into 85mm petri dishes. Let the agar harden.
X-Gal (2ml) Store at 4°C for up to 1 month or at room temperature for
100mg 5-bromo-4-chloro-3- up to 1 week.
indolyl-β-d-galactoside
LB plates with ampicillin/IPTG/X-Gal
Dissolve in 2ml N,N´-dimethyl-formamide. Cover
with aluminum foil and store at –20°C. Make the LB plates with ampicillin as above; then
supplement with 0.5mM IPTG and 80µg/ml X-Gal and
LB medium (per liter) pour the plates. Alternatively, 100µl of 100mM IPTG and
10g Bacto®-tryptone 20µl of 50mg/ml X-Gal may be spread over the surface
5g Bacto®-yeast extract of an LB-ampicillin plate and allowed to absorb for
5g NaCl 30 minutes at 37°C prior to use.
Adjust pH to 7.0 with NaOH.
SOC medium (100ml) 2X Rapid Ligation Buffer, T4 DNA Ligase (provided)
2.0g Bacto®-tryptone 60mM Tris-HCl (pH 7.8)
0.5g Bacto®-yeast extract 20mM MgCl2
1ml 1M NaCl 20mM DTT
0.25ml 1M KCl 2mM ATP
1ml 2M Mg2+ stock, filter-sterilized 10% polyethylene glycol (MW8000, ACS Grade)
1ml 2M glucose, filter-sterilized Store in single-use aliquots at –20°C. Avoid multiple
Add Bacto®-tryptone, Bacto®-yeast extract, NaCl freeze-thaw cycles.
and KCl to 97ml distilled water. Stir to dissolve.
TYP broth (per liter)
Autoclave and cool to room temperature. Add 2M
Mg2+ stock and 2M glucose, each to a final concen- 16g Bacto®-tryptone
tration of 20mM. Bring to 100ml with sterile, 16g Bacto®-yeast extract
distilled water. The final pH should be 7.0. 5g NaCl
2.5g K2HPO4
2M Mg2+ stock
20.33g MgCl2 • 6H2O
24.65g MgSO4 • 7H2O
Add distilled water to 100ml. Filter sterilize.
10.D.Related Products
PCR Cloning Systems
Product Size Cat.#

pTargeT™ Mammalian Expression Vector System 20 reactions A1410
Direct mammalian expression from a T-Vector.
Amplification Products
A partial list of our amplification products is given on the next page. Please visit our Web site at:
www.promega.com/applications/pcr/ to see a complete list.
10.D. Related Products (continued)
Thermostable DNA Polymerases
Product Size Cat.#

GoTaq® Hot Start Polymerase 100u M5001
GoTaq® DNA Polymerase 100u M31711, M30012
GoTaq® Flexi DNA Polymerase
(allows optimization of Mg2+ concentration in reaction) 100u M83011, M82912
Additional sizes available.
1
Cat.# M3171 & M8301 are available in Europe or through Distributors supported by Promega European Branch Offices.
2
Cat.# M3001 & M8291 are available in all other countries, including the United States.
PCR Master Mixes
Product Size Cat.#

GoTaq® Hot Start Green Master Mix 100 reactions M5122
1,000 reactions M5123
GoTaq® Hot Start Colorless Master Mix 100 reactions M5132
1,000 reactions M5133
GoTaq® Green Master Mix 100 reactions M71121, M71222
1,000 reactions M71131, M71232
GoTaq® Colorless Master Mix 100 reactions M71421, M71322
1,000 reactions M71431, M71332
GoTaq® Master Mixes are premixed solutions containing GoTaq® DNA Polymerase, GoTaq® Reaction Buffer (Green or
Colorless), dNTPs and Mg2+.
1
Cat.# M7112, M7113, M7142 & M7143 are available in Europe or through Distributors supported by Promega European
Branch Offices.
2
Cat.# M7122, M7123, M7132 & M7133 are available in all other countries, including the United States.
PCR Purification Systems
Product Size Cat.#

Wizard SV Gel and PCR Clean-Up System
®
50 preps A9281
250 preps A9282
Wizard SV 96 PCR Clean-Up System
®
1 × 96 preps A9340
Additional sizes available.
Competent Cells
Product Size Cat.#

JM109 Competent Cells, >108cfu/µg 5 × 200µl L2001
Single Step (KRX) Competent Cells 20 × 50µl L3002
RT-PCR Systems
Product Size Cat.#

Access RT-PCR System 100 reactions A1250
AccessQuick™ RT-PCR System 100 reactions A1702
500 reactions A1703
ImProm-II™ Reverse Transcription System 100 reactions A3800
Available in additional sizes.
dNTPs
Product Size Cat.#

PCR Nucleotide Mix (10mM each) 200µl C1141
1,000µl C1145
dATP, dCTP, dGTP, dTTP, each at 100mM 10µmol of each U1330
Accessory Products
Product Size Cat.#

X-Gal 100mg (50mg/ml) V3941
IPTG, Dioxane-Free 1g V3955
5g V3951
10.E. Summary of Changes

The following changes were made to the 6/15 revision of this document:
1. Removed BstZI Promega catalog number and added BstZI isoschizomers to Notes in Sections 5.B and 5.D. Also
unbolded BstZI in Tables 3 and 5.
2. Removed expired license statements.
© 1998, 1999, 2003, 2005, 2007, 2009, 2010, 2015 Promega Corporation. All Rights Reserved.
GoTaq, pGEM and Wizard are registered trademarks of Promega Corporation. AccessQuick, ImProm-II, pTargeT and PureYield are trademarks of
Promega Corporation.
AmpliTaq is a registered trademark of Roche Molecular Systems, Inc. Bacto is a registered trademark of Difco Laboratories. DNASTAR is a registered
trademark of DNASTAR, Inc. Falcon is a trademark of Becton, Dickinson and Company. GenBank is a registered trademark of the U.S. Dept. of Health
and Human Services. SURE is a registered trademark of Stratagene. Vent and Deep Vent are registered trademarks of New England Biolabs, Inc. XL10
Gold is a registered trademark of Stratagene.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date
information on Promega products.

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TECHNICAL MANUAL

pGEM®-T and pGEM®-T

2. Product Components and Storage Conditions......................................................................................... 3

6 General Considerations for PCR Cloning.............................................................................................. 12

1.A. Vector Features

1.B. Important Considerations for Successful T-Vector Cloning

pGEM®-T Vector System I 20 reactions A3600

pGEM®-T Vector System II 20 reactions A3610

pGEM®-T Easy Vector System I 20 reactions A1360

pGEM®-T Easy Vector System II 20 reactions A1380

3.A. Ligation Protocol

Standard Positive Background

3.B. Optimizing Insert:Vector Molar Ratios

ng of vector × kb size of insert

50ng vector × 0.5kb insert 3

4.A. Transformation Protocol

Materials to Be Supplied by the User

4.B. Example of Transformation Efficiency Calculation

5.A. Sequence and Multi-Cloning Site of the pGEM®-T Vector

CCATG GCCGC GGGATT3′

NcoI SacII SpeI NotI PstI SalI NdeI

pGEM®-T Vector sequence reference points:

GCGGC CGCGG GAATT CGATT3′

SP6 Transcription Start

pGEM®-T Easy Vector sequence reference points:

T7 RNA polymerase transcription initiation site 1

6.A. PCR Product Purity

6.B. Properties of Various Thermostable Polymerases

Table 1. Comparison of PCR Product Properties for Thermostable DNA Polymerases.

Thermostable DNA Polymerase

5´→ 3´exonuclease activity Yes Yes Yes No No No No

3´→ 5´exonuclease activity No No No Yes Yes Yes Yes

Table 2. Comparison of A-Tailing Procedures.

DNA Purification System prior to A-tailing.

Start with 1–7µl of purified PCR fragment generated by a

Add dATP to a final concentration of 0.2mM.

Add 5 units of Taq DNA Polymerase.

Add deionized water to a final reaction volume of 10µl.

Incubate at 70°C for 15–30 minutes.

Use 1–2µl in a ligation reaction with

Symptoms Causes and Comments

Symptoms Causes and Comments

10.A. pGEM®-T Vector Restriction Enzyme Sites

Enzyme # of Sites Location Enzyme # of Sites Location

Table 4. Restriction Enzymes That Do Not Cut the pGEM®-T Vector.

AccB7I BbsI BstEII FseI PinAI SplI

10.B.pGEM®-T Easy Vector Restriction Enzyme Sites

Enzyme # of Sites Location Enzyme # of Sites Location

AccB7I BbsI BstEII HindIII PmeI SplI

10.C. Composition of Buffers and Solutions

IPTG stock solution (0.1M) LB plates with ampicillin

Adjust pH to 7.0 with NaOH.

PCR Cloning Systems

Product Size Cat.#

Thermostable DNA Polymerases

Product Size Cat.#

PCR Master Mixes

Product Size Cat.#

PCR Purification Systems