8 Plasmid Cloning

Procedures You
Need to Know

And Some Tips for Mastering Them

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now
8 Plasmid Cloning Procedures You Need to Know (And Some Tips for Mastering
Them)
© 2012 BioData Ltd. All rights reserved.

Content may be copied for personal use. It may not be copied or distributed for
other purposes without prior written consent from BioData Ltd.

Copies may be acquired by downloading from http://lp.labguru.com/plasmid-

cloning-procedures-you-need-to-know/

Thank you for downloading 8 Plasmid Cloning Procedures You

Need to Know (And Some Tips for Mastering Them) from
Labguru.com.

Labguru creates digital tools for increasing the produc2vity of

academic and industrial laboratories. Our web‐based so;ware is
currently used by thousands of researchers in hundreds of labs
worldwide. It is available FREE for personal use. To get started, check
out www.labguru.com.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now
 8 Plasmid Cloning Procedures
You Need to Know
And Some Tips for Mastering Them

The basic procedure performed by any molecular biologist is, naturally, cloning. You
may wish to clone a DNA fragment into a plasmid for many purposes, such as protein
expression and purification, gene knock out / silencing / overexpression, reporter
gene fusion to a gene of interest, and many more. Whether you plan on doing one
thing or another with the plasmid you cloned, when it comes to the cloning
procedure you will need to perform the same steps, which are the ABC’s of
molecular biology.

However simple it may seem, when you don’t have enough experience in cloning
plasmids, you can find yourself struggling endlessly, going over the same protocol
over and over again for weeks and even months, gaining no success or insights as to
what went wrong. I’ve seen so many frustrated graduate students and postdocs
walking around the corridors of research buildings, knocking on neighboring lab
doors looking desperately for help and advice. I certainly do not claim to be a
cloning guru, on the contrary. I’ve had my fair share of nightmare-haunted nights,
full of dreams featuring linear vectors unwilling to circulate, and images of me in
front of the PhD committee having nothing to discuss. It took me quite a while (and
many frustrations and disappointments) to realize that before jumping into the icy
cold water of molecular biology protocols, I need to thoroughly read, learn and
consult experts. It’s as simple as that: the more you know about the cloning
protocols, understand the rationale behind each step, and the function of each
component used, the faster and easier you’ll cross the bridge from preparing the
molecular tools for your research to the actual science making.

We therefore prepared here for you, the molecular biologist, a step-by-step protocol
for cloning plasmids. For each step you’ll find some useful suggestions and
recommendations. The procedures discussed in this protocol include:
• Polymerase chain reaction
• Ligation
• Bacterial transformation
• Plasmid DNA isolation
• And more
You are invited to download and use the protocol as a basic guide to your cloning.

Good luck in your cloning!

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 3
1) Plan your cloning strategy and arrange your data
Begin with data collecting - make sure you have a very clear plan of
• What your insert DNA fragment is going to be
• Which plasmid vector you‘ll use as recipient of your insert
• Their complete sequences, and
• What restriction enzyme you’ll use at each end of the insert.

Based on my experience, good planning leads to fast and successful cloning. You do
not need to reinvent the wheel. Most probably, someone in your lab has already
cloned the same insert you are about to. If your lab is organized this should not be
too difficult or time consuming. Simply go to whatever management system your lab
uses, and search for possible origins for your insert. Make a list of all the available
vectors bearing that insert, and their locations in stock boxes. Prior to getting back
to the bench don’t forget to consult the person who cloned the vector you’re about
to use, for specific advice on practical aspects of working with this vector. The
experience of your lab-mates is invaluable!

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 4
2) Polymerase Chain Reaction – PCR
The best way for obtaining large amounts of your desired insert is, naturally, using
PCR.

a) Start your PCR planning by carefully designing your primers. Correct primer
design is key factor for a successful PCR that yields the desired product. Don’t
forget to include the relevant restriction site on both primer edges fitting the
orientation of cloning into the recipient vector. If possible, use a different
restriction enzyme at each edge, but try choosing two enzymes that can digest in
the same buffer, to save time by performing a double digest. When you design
your primers pay attention to their sequence at the 5’ end; you need to add a
few extra nucleotides that precede the restriction site you planted at the 5’ end
in order to reach higher percentages of PCR product digestion. Determine those
extra nucleotides according to the manufacturer's recommendation for each
enzyme. Once you have finished editing the primers’ sequences, it is very
important that you also make sure there are no secondary structures, or primer
dimers, and that the Tm of both primers is similar. Don't forget that, since in
most cases, the 5’ end of your primer is not complementary to the sequence
amplified due to the restriction site planted, the Tm calculation should not
include the non-complementary nucleotides. There are many primer calculation
tools available on the Internet for free. As someone who performed PCRs galore,
I advise you, do not skip this test. You’ll find that if the designed primers were
not optimized for the reaction intended for them, you end up wasting precious
time, materials and money, but you do not get your desired PCR product.

b) Don’t use just any Taq polymerase present in the lab. If your PCR is meant to
amplify a product that will serve for expression, it is necessary to use a
proofreading enzyme. Also, verify that the Taq you use is capable of efficiently
amplifying the length of your product, some Taq polymerases cannot synthesize
more than 1-2 Kb.

(Continued next page…)

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 5
2) Polymerase Chain Reaction – PCR
(…Continued)

c) Include as many controls as possible in the reaction. Preparing a few more

reactions will eventually save you a lot of time and effort when, trying to
decipher your results, you’ll find yourself straining to figure out what went
wrong, why you see no bands in the gel, or what are the extra mysterious bands
you do see that are of unexpected length. I recommend for each PCR a “no
template reaction” for each primer pair, and at least one amplification of a
positive control product, to check that your PCR mix works well.

d) Arrange and organize ahead all the ingredients for your reaction, and mark your
tubes so that it is clear what is to be added into each. Make a list of the
ingredients, calculate the volumes to be added of each, and make time for
preparing and running the reaction. Programme the PCR cycler prior to mixing all
the components of the reaction. It may seem as if you can put it together all in 5
minutes in between experiments, but, believe me, if you are not focused on the
preparation and mixing of all the components in the correct volumes and order,
you’ll soon enough be asking yourself questions such as: did I add the dNTPs to
the mix tube? was that primer diluted already? did I add the positive control
template? why do I have 100 microliters left in my mix tube even though all
sample tubes seem full?

e) After the reaction is done, load a small volume of each sample on agarose gel and
check if it went well. Don’t forget to document! You might want to repeat this
protocol or change it a bit. If you have an annotated picture of the gel next to
reaction list and PCR program you won’t need to trust your memory in a few
months, when you’ll need to retract your steps or repeat them.

f) Great! now you have a tube containing your desired insert amplified to a large
amount, almost ready for cloning. It is highly recommended to be rid of the PCR
template prior to using the insert for downstream enzymatic reactions. You can
either perform gel purification using a purification column, or directly apply the
reaction onto a PCR product purification column. These columns are
commercially available.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 6
3) Restriction
The next step is to digest both the insert (your PCR product) and the recipient
plasmid you chose.

a) Carefully read the manuals provided with the restriction enzymes you are about
to use! Make sure to use the best fitting buffer for a double digest, and to
incubate at the optimal temperature. If a sequential digestion is required,
consult the recommendations of the supplier on how to perform it.

b) Remember that the definition of one enzyme unit is the number of enzyme
molecules that fully digest 1 microgram of DNA in 1 hour, and calculate the
number of enzyme units required according to the amount of DNA you put in the
digestion reaction.

c) No matter how much DNA you put into your reaction it is crucial that the volume
of the enzyme added will not exceed 10% of the total reaction volume. This is
because of the high glycerol concentration in the enzyme solution. The total
glycerol concentration in the reaction should not exceed 5%, or it will inhibit the
digestion.

d) It is also highly important that you do not over incubate the reactions. Most of
the supplied enzymes today will digest a large amount of DNA within 1 hour if
placed in optimal buffer and temperature. Check if the enzymes you use posses
star activity. Such activity means that if incubated longer than the recommended
period, an enzyme might non-specifically digest DNA sequences other then its
recognition site. It is therefore important to consult the manufacturer's protocol,
and incubate in accordance.

e) To shorten the incubation period you can use the “fast-digest” enzymes supplied
by some companies. These are guaranteed to fully digest 1 microgram of DNA in 5
minutes, so you will not need to worry about star activity, but keep in mind these
enzymes are costly.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 7
4) Ligation
Following restriction enzyme digestion of both your insert and the vector you can
perform the ligation reaction.

a) Insert - Usually, if the restriction enzymes and ligase were all provided by the
same manufacturer, you will not even need to purify your insert from the
restriction reaction, but make sure you have inactivated the restriction enzymes
prior to ligation. Information on how to inactivate each restriction enzyme is
provided in the suppliers manual. In case inactivation is impossible, or that the
restriction buffer is not suitable for the ligase you’re using, you’ll need to load
the restriction reaction onto a purification column to separate the insert from the
restriction enzymes.

b) Plasmid - If the digestion has removed a fragment from your plasmid it is crucial
that you purify your plasmid to get rid of that fragment, as it will compete your
desired insert during ligation. The best way to do that is by purification of the
linear from gel. Alternatively, if you’re running low on plasmid amounts, you can
simply add a third restriction enzyme that does not cut within your plasmid, but
has a recognition site in the fragment you wish to be rid of. This will eliminate
competition by the fragment excised, and will minimize loss of precious material.
Of course, you’ll need to inactivate all 3 enzymes used before you go on with the
ligation reaction.

c) Ligation - The reaction is very simple to combine and includes buffer, plasmid,
insert and the ligase. The most important consideration here is the molar ratio
vector : insert. Typically, a ratio of 1:3 works well (3 insert molecules per 1
vector molecule). You’ll need to measure the concentration of the vector and
insert and combine them accordingly. Always read carefully the manufacturer’s
supplied manual of your ligation mix prior to performing the reaction, to know
the exact incubation conditions.

d) Control - If you have enough material it is highly recommended to perform a “self

ligation” reaction of your vector only with no insert added. This will help you get
an idea of the efficiency of insert-ligation versus plasmid re-circularization before
you start analyzing bacterial colonies and find out a few days later you have no
insert in the plasmids you obtained.

(Continued next page…)

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 8
4) Ligation
(…Continued)

e) To make sure your ligation worked prior to moving on with the bacterial
transformation it is recommended to load a small volume of the reaction on a gel
next to the linear plasmid mixed with the insert, but with no ligase added.
Positive result for ligation should be displayed as bands of various lengths on the
gel, indicating that your ligase is intact, and managed to connect the DNA
fragments in your ligation solution with one another; each band on the gel
containing a different number of ligated fragments.

f) Finally, if you have unsuccessfully performed the same ligation over and over
again, try dephosphorylating your vector before moving on to ligation in order to
eliminate the possibility of self-ligation. Another tip for increasing ligation
efficiency is to heat inactivate the ligase prior to transforming the ligated
plasmid into bacteria.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 9
5) Bacterial Transformation
After performing the ligation reaction, you need to deliver the plasmid DNA into host
bacterial cells for massive production.

At this point you need to consider two factors:

1. Which strain of bacteria to use, and
2. What type of transformation technique to conduct.

Bacteria: Note that there are many bacterial strains that have been engineered for
various aims. You should therefore choose those for cloning and maintenance
purposes. The strains DH5-alpha, XL-1 blue or JM109 will serve you well (you can
view a complete list of E. coli strains here). It is critical to know this before hand, as
transformation requires the preparation of bacterial cells before the introduction of
your ligation (see below).

Technique: You can use electroporation, which is known to be more efficient by

about three orders of magnitude in comparison to the heat shock transformation
method, but requires dedicated electroporator and special corvettes. The heat shock
method only requires a wet or dry heater block set to 42 degrees Celsius, which is
commonly found in molecular biology labs. Take into consideration that each
technique requires a different preparation (or purchase) of your choice competent
strain. Opt for the electroporation technique if you can, especially if you routinely
find your LB plate empty of colonies.

Following transformation - incubate your plates overnight at a 37OC incubator.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 10
6) Clone Verification
Good morning!
Open the incubator’s door, and look at your plates.....many bacterial colonies? That’s
great! Most probably this means your ligation worked, unless you have a similar
number of colonies on your self ligation plate. If you don’t, the next step for you is
to look for positive colonies, containing your plasmid. You don’t need to check more
than 15 colonies if on your self ligation plate you have much less colonies than on
your ligation plate.

A common way of performing this test is colony PCR, for amplification of your insert
directly from lysed bacterial cells containing the plasmid you cloned it into. In this
procedure you perform a regular PCR using bacterial colonies as templates. Include
in the reaction either the primers used to amplify the insert, or primers
complementary to plasmid sequences on both ends of the insert:
• First, prepare a Petri dish marked with numbers 1-15 (for each colony you’ll
check). The plate should contain the same medium used for yesterday’s plating -
don’t forget the antibiotics.
• Next, prepare the PCR mix (that should include all reaction components except for
the template) and divide it to pre-marked 1-15 reaction tubes. Mark control tubes
as well: positive and negative control. Use a sterile tip to very gently touch one
colony from the ligation plate, then, prick with this tip very gently the Petri dish
next to the number 1 marked on it.
• Now, insert the tip into PCR tube marked 1, and slightly mix the solution with the
tip. Discard the tip. The bacteria on the tip serve as your template #1 in this
reaction. Perform the same procedure with 14 other colonies. Make sure to touch
one colony exclusively each time, and don’t forget to incubate the Petri dish with
the 15 colonies analyzed to allow colony growth.
• As negative control template use one colony from the self ligation plate, and as
positive control template use the plasmid (or genomic DNA) from which you
derived your insert. Include other controls as elaborated under the PCR section.
Perform the reaction on the thermo cycler pre-programed, but don’t forget to
perform a first step of 5 minutes at 95OC. This step is necessary for lysing the
bacterial cells, allowing your plasmid (the template) to get into the reaction mix.
• Once the reaction is done analyze the products on an agarose gel, and mark the
colonies from which the insert was successfully amplified - you won’t need more
than 2 from which to isolate your plasmid. Prepare a small volume liquid culture
of the 2 colonies you chose by using colonies that grew on the Petri dish marked
1-15. Don’t forget to add antibiotics to your liquid cultures, and grow them
overnight.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 11
7) Plasmid DNA Isolation
You are one protocol away now from finally having your desired plasmid!

Plasmid isolation these days is mostly performed using commercial columns kits of
mini/midi/maxi preps. For your purposes, a mini-prep kit will do the trick. You will
need to grow your potential positive colonies overnight in 5ml LB (supplemented
with appropriate antibiotics) and the next day start the mini-prep protocol according
to the manufacturers recommendation. Although these kits are very easy and simple
to use you may encounter low yield issues or contaminated plasmid DNA with
proteins or genomic DNA. Usually low yield issues are related to either of the
following:

1. Low pellet mass - bacteria was not grown to high enough O.D or bacterial lysis
due to phage contamination.
2. Inefficient lysis of bacterial cells - this can be caused by inefficient resuspension
of bacterial pellet with the hypotonic solution (the first solution). Using medium-
speed centrifuge force (6000g) when centrifuging the overnight bacterial
inoculum leads to a more loose pellet which is easier to resuspend.
3. Old washing solution / ethanol was not added to the supplier’s washing solution -
washing steps between binding and elution of the DNA prep is performed with
high concentration of ethanol (>70%). The supplied washing solution should be
supplemented with absolute ethanol by the users so make sure you’re using a
ready-to-use solution. In addition, over time ethanol may evaporate, and thus it’s
concentration may decrease, leading to partial elution of your bound plasmid.

(Continued next page…)

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 12
7) Plasmid DNA Isolation
(…Continued)

Protein contamination (reads of 260nm/280nm<1.8) is usually related to insufficient

wash steps following the binding of your DNA to the column or to inclusion of
particles from the pellet following the cold centrifuge step (Maximum speed for 10
minutes at 4 degrees Celsius). When removing the supernatant from the tube make
sure not to touch or include any white particle of the pellet, which contains mainly
membrane and protein fraction of the whole bacterial lysate.

Salt contamination (reads of 260nm/230nm<1.5) can be the result of precipitation

step and might ruin any sequencing effort. Thus, it is recommended to use two wash
steps with the wash buffer, making sure to wash even the rim of the spin column.

Submit your DNA sample to sequencing with the appropriate primer, either forward
or reverse - if your sequence is long (over 800bp) you might need to sequence it both
from the forward and from the reverse side, due to the size limitations of the
sequencing technique. You should also take into consideration the fact that the first
sequenced ~10-30 bp might be hard to interpret, so you might need a different
primer.

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 13
8) Make sure you have cloned the desired plasmid
When you receive your sequence file it will most probably be with the extension
of .ab1. You can open such a file with Bioedit (http://www.mbio.ncsu.edu/
bioedit/bioedit.html) or GENtle (http://gentle.magnusmanske.de/) programs -
both are freely available.

Once you’ve got the sequence opened on your monitor take a look at the
chromatogram - if the peaks are flat or not well resolved, your sequencing effort
wasn’t successful. See http://seqcore.brcf.med.umich.edu/doc/dnaseq/
interpret.html for a detailed description on how to analyze sequencing results.

If you get a good sequence, now’s the time to check if whole effort worked. The
quickest way to do that is to BLAST it through NCBI’s website1. Scan the top results:
if you don’t see your gene, than you got a self-ligated vector result. Don’t throw
away that result yet! You can still search for the ligation point and possibly analyze
what might be the cause of the self ligation (maybe only one digestion occurred?).

If you do find your gene, don’t pop that champagne just yet - scan the sequence well
for mutations. Ask a labmate to help you as it is terribly easy to miss a mutation or
two.

Sequence is 100% identical without any gaps? Congrats, you’ve just successfully
cloned your gene!

Found a mismatch? Don’t worry just yet - go back to the sequence chromatogram
and identify the position in which this mutation is observed. How does the peak look?
Is it an overlap of a neighboring peak or does it look fine? If the peak looks good than
you’ve got yourself a ligated mutated gene but you should translate your gene and
see if it might be a null mutation.

Cloning a gene is an arduous effort with lots of challenges, but if you do this for
some time you will quickly realize that your efficiency and success rise with time.
Don’t loose heart in the face of failures - they happen to everyone! Learn from them
and clone away again until you get it done!

Good luck!

1. http://blast.ncbi.nlm.nih.gov/Blast.cgi?
PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=&LINK_LOC=blasttab&LAST_PAGE=blastp

Plan. Track. Get Results.

www.labguru.com
Do More Science. Free for Personal Use – Try it Now 14