KT60 GFP Cloning Teaching Kit
KT60 GFP Cloning Teaching Kit
KT60 GFP Cloning Teaching Kit
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CONTENTS
Page No.
v Objective 1
v Principle 1
v Kit Description 5
v Materials Provided 7
v Procedure 9
v Appendix 14
v Ordering Information 15
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Objective:
To understand the basic concept of cloning and perform
some of the steps involved, using the GFP gene (Green
Fluorescent Protein).
Principle:
Molecular cloning or gene cloning involves insertion of a
DNA fragment (gene of interest) into a cloning vector. The
recombinant vector is subsequently transformed into a
suitable host to generate the desired clones.
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• Vector DNA: Cloning vectors are circular DNA vector as 5’ phosphate groups of the insert are intact and
molecules in which DNA fragments/insert are maintained thus efficiency of insert ligation to the vector increases.
and amplified. Commonly used cloning vectors include: Another method employed to prevent self ligation of vector is
Plasmids, Cosmids, Phagemids and Yeast artificial to cut the DNA with two different restriction enzymes resulting
chromosomes etc. in mismatched ends that cannot ligate.
Vectors have the following features: Joining of DNA: Once the insert and vector DNA have
o An origin of replication recognized by the host cell been isolated and cut with specific restriction enzyme(s),
replication machinery. they must be ligated. DNA ligase is the glue of molecular
o A selection marker that enables the identification of genetics that holds the DNA together by creating a
transformants and/or transfectants. phosphodiester bond between the two DNA ends to create a
o One or multiple restriction endonuclease recognition recombinant DNA molecule. When a single restriction
sites that allow the insertion of the gene of interest. enzyme is used to cut the vector and insert DNA, ligation
occurs in either direction/orientation. However, the insert can
Vector DNA purification protocols vary depending upon be forced to ligate to the vector in the desired orientation, by
the type of vector chosen for cloning experiments. Smaller using two different restriction enzymes to cut both the vector
sized cloning vectors like plasmids, cosmids and phagemids and insert DNA.
are easier to purify and are usually done by alkaline lysis
method and subsequent phenol/chloroform extractions. Amplifying the recombinant DNA: The recombinant
DNA thus created is introduced into a compatible host such
Cutting of DNA: In order to carry out recombination that the vector DNA is replicated resulting in amplification of
between the vector and the insert DNA, it is necessary to the insert DNA. When plasmid DNA is introduced into host
specifically and reproducibly cleave the DNA. Restriction cell, the process is referred to as transformation and in
enzymes are the scissors of molecular genetics, which case of viral DNA the process is transduction. A successful
recognize specific nucleotide sequences and cut the DNA at vector DNA transfer is monitored by selecting for the genetic
these points to generate sticky or blunt ends. Generally, the marker present on the vector. For eg., plasmid vectors usually
same restriction enzyme is used to cleave both the insert have dominant selectable markers such as antibiotic
and vector DNA. Following restriction enzyme digestion of resistance genes that confer growth on media containing
the vector, the DNA is dephosphorylated i.e., 5’ phosphate appropriate concentration of an antibiotic. In case of viral
group is removed by treating with alkaline phosphatase. This vectors, one observes for formation of plaques.
is done to reduce self ligation of vector molecules by T4 DNA
ligase. However, this does not prevent ligation of insert to the
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Screening for clones: The final step involved in cloning Kit Description:
is the means to detect the right clone among a population of
In this kit, a vector and an insert DNA (GFP gene isolated
colonies or plaques. This screening mechanism will depend
from a bioluminescent jelly fish Aequorea victoria) are provided
upon the vector chosen as the cloning vehicle. For example,
along with the host (E. coli strain DH5α). Using the kit
pBR322 (plasmid vector) carries genes for ampicillin
students will carry out the following experiments:
resistance and tetracycline resistance. Both these genes
have sites for restriction enzymes that allow for cloning and
• Ligation: GFP gene will be ligated to the vector using
selection of recombinants by “insertional inactivation” of one
the enzyme T4 DNA ligase at 16°C. Since, the vector
of the genes. Hence, cells with vector alone will be resistant
and insert DNA have been cleaved with the same single
to both the antibiotics, but cells with recombinant DNA will
restriction enzyme, ligation of insert to vector will occur
be resistant to only one antibiotic and can be selected by
in either direction/orientation.
replica plating.
• Preparation of competent cells: Host E.coli cells
will be made competent by using a solution having salts
like calcium chloride, magnesium chloride, manganese
chloride, etc.
• Transformation: The ligated sample will then be
transformed into competent E.coli cells and plated on
LB plates containing ampicillin. Cells having
recombinant DNA or self ligated vector DNA will grow
since the plasmid carrying ampicillin resistance gene
confers antibiotic resistance to the host cells.
• Screening: The plates will then be visualized under
UV light to screen for clones. This is because, the
unique 3 dimensional conformation of the GFP releases
energy in the form of visible green light on exposure to
UV light. Hence, only cells having the insert in the right
orientation will glow and these are the clones. However,
there will be other clones where in the insert is ligated
in the reverse orientation resulting in no expression of
the protein. These clones will not glow on exposure to
UV light.
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Materials Required:
Equipments : Water bath/Dry bath, Centrifuge
(preferably refrigerated),
UV transilluminator (312 nm),
Spectrophotometer.
Glasswares : Conical flask, Petri plates, Test tubes.
Reagent : Distilled water.
Other Requirements : Crushed ice, Cuvette (of 1 cm path
length), Micropipette, Micro Tips,
Thermometer, Centrifuge tubes, .
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Note: Procedure:
• Read the entire procedure prior to starting the Day 1: Revival of Host
experiment. 1. Break open the lyophilized Host vial. Add 0.1 ml of LB
• Ensure that all the required components are ready prior broth.
to starting the experiment. 2. Streak a loopful of the suspension onto LB plate
• All microbiological procedures should be done under (in duplicates).
aseptic conditions. 3. Incubate the plates at 37°C, overnight.
• Revive the strain as soon as the lyophilized vial is
opened. Ligation of Vector to Insert
• Prepare competent cells within 3 days of reviving the 4. Thaw the Ligase Assay Buffer, vector and Insert DNA.
strain. Note: Thaw the Ligase Assay Buffer vial on ice,
• Entire procedure of preparing competent cells should store at -20°C immediately after use.
be done under aseptic conditions. 5. Set up ligation reaction as indicated in table 1:
• Carry out transformation as soon as the competent cells Table1:
are prepared. Reagent Amount
• Storage of competent cells may result in poor/no Water 11 µl
transformants.
Vector DNA 2 µl
• Transformation efficiency of competent cells should be
more than 1 x 105 µg of DNA. Efficiency lower than this Insert DNA 4 µl
may lead to lower number of transformants. Ligase assay buffer 2 µl
• Pre-cooled tubes, pipettes, centrifuge tubes and T4 DNA ligase 1 µl
Solution A, prior to preparation of competent cells.
• Solution A supplied is sterile, handle under aseptic Mix the contents by tapping gently and incubate at
conditions. 16°C waterbath, overnight.
• 5 ligation reactions and transformations are to be done Note: Set up five ligation reactions simultaneously.
simultaneously.
• For preparation of media, antibiotic, etc., refer appendix Day 2: Preparation of Competent Cells
on page 16. 6. Pick 10-12 moderate sized colonies from the LB plate
and innoculate into 100 ml of LB broth (in a 1 litre
conical flask).
7. Incubate at 37°C, in a shaker. Grow until OD A 600
reaches 0.3. This takes about 2-3 hours.
8. Chill the culture flask on ice for 10-20 minutes.
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9. Transfer the culture aseptically into sterile centrifuge 18. Label the remaining 200 µl of competent cells as non-
tubes and centrifuge at 6000 rpm for 8 minutes, at 4°C transformed cells, place on ice till the plating step (step
or at Room temperature (RT). 24).
10. Discard the supernatant. 19. Tap all the vials gently and incubate on ice for
11. Resuspend the cell pellet very gently in 2 ml of ice-cold 20 minutes.
solution A , using a pre-chilled pipette. Care must be 20. Heat shock the cells by placing the vial(s) of ligated
taken not to remove the tubes from ice during sample and Control DNA in 42°C water bath for
resuspension. Then add 33 ml of solution A and 2 minutes, then return the vials to ice and chill for
resuspend gently. 5 minutes.
12. Keep the centrifuge tubes on ice for 20 minutes. 21. Add 0.5 ml of LB broth aseptically to the vial(s) and
Centrifuge at 3500 rpm for 15 minutes at 4°C or at RT. incubate at 37°C (in a shaker) for an hour. This is to
13. Discard the supernatant and chill the tube on ice. allow the bacteria to recover and express the protein.
Resuspend the pellet in 3 ml of ice-cold solution A. 22. Pipette 200 µl from each of the vials transformed with
the ligated mix onto LB-Amp plates and spread
Note: Resuspension should to be done gently as
thoroughly using a spreader or pipette.
the cells are very fragile at this stage.
23. Pipette 200 µl of LB onto a LB -Amp plate and spread
14. Aliquot 0.6 ml of the above suspension into 5 different 20 µl of the competent cells transformed with Control
pre-chilled 1.5 ml vials aseptically. DNA. Label this as ‘Control Plate’.
15. Heat inactivate the ligated samples at 65°C for 24. Plate 200 µl of non-transformed cells onto another
10 minutes. Centrifuge at 5000 rpm for 2 minutes and LB-Amp plate to check for contamination. Label this
keep the vials on ice. as ‘Non-Transformed Plate’.
16. Aliquot 2 µl (10 ng) of Control DNA into each of 5 25. Incubate the plates at 37°C, overnight.
pre-chilled 1.5 ml vials. 26. Observe the plates under UV-light (312 nm).
Note: Intensity of the glow could vary if the plates
Transformation: are observed at 254 nm.
Note: Use single aliquot of competent cells 27. Count the number of colonies on the plate and calculate
(0.6 ml) for one set of transformation experiment. the Transformation Efficiency.
17. Take one vial of ligated sample (Day 2 Step 15) and Note: Steps 17 to 27 explains the protocol for one
Control DNA (Day 2 Step 16). To each of these add set of transformation experiment. Carry out 5 such
200 µl of competent cells. Tap the vials gently and transformations simultaneously.
incubate on ice for 20 minutes.
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Notes:
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