Epicentre's Scientists frequently need to clone and express genes as
part of their R&D projects. These genes may be cloned directly from
restriction enzyme-digested genomic DNA or cDNA or, if the gene sequence
is known, the insert may be obtained by PCR amplification using genomic
DNA or cDNA as a template. Whatever the source of the insert, they believe
the following tips will enable you to efficiently and successfully clone
Tip # 1. Don't expose your insert to UV light.
UV transilluminators have been used for many years for visualizing DNA
bands separated on agarose electrophoresis gels. Damage to DNA by the
UV light is enough to be detectable after even 5 seconds of exposure
and enough to make certain regions unreadable on a sequencing gel within
60 seconds. One solution to avoid exposure of the gel-separated DNA
to UV is to run an extra duplicate lane next to the lane with the fragment
to be cloned. The duplicate lane is cut out of the gel, stained and
visualized on the transilluminator and then used as a marker for where
to cut out the band from the lane that is not exposed to UV. A better
solution is to use a Dark Reader™ Visible Light Transilluminator
from Clare Chemical Company. A Dark Reader uses only visible light,
which does not damage DNA and is not harmful to people, and two filters.
The gel is first exposed to light that has passed through a blue filter.
It filters out all wavelengths except the excitation wavelength of
the intercalator dye. Then, the bands are viewed through an orange
filter, which only allows the excitation wavelengths to pass through.
Sensitivity using SYBR Gold or other dyes exceeds UV light with ethidium
bromide. More important, exposure to Dark Reader light does not affect
cloning efficiency. In one experiment at Epicentre, a 30-second exposure
of phage T7 DNA to UV light on a transilluminator decreased the number
of transformants obtained using that DNA as an insert by more than
two orders of magnitude, but a 5-minute exposure to Dark Reader light
had no effect on cloning efficiency. See www.clarechemical.com for
Tip # 2. Minimize losses of your insert DNA.
If you purify your insert DNA on an agarose gel, you will obtain quantitative
recoveries by using a low-melting-point agarose gel and then digesting
the gel slice containing insert band using GELase™ Agarose Gel-Digesting
Preparation. Alternatively, if you're making the insert by PCR, you
can probably skip the gel and remove the nucleotides and primers by
precipitating the PCR product using the DNA Fragment 2X Precipitation
Tip # 3. If you generate an insert by RT-PCR, don't
take chances by using the wrong PCR enzyme or conditions.
Avoid any PCR enzyme or reaction that uses manganese cations. For example,
our MasterAmp™ RT-PCR Kit for High Sensitivity is optimized for
and delivers the claimed highest detection sensitivity, but it uses manganese
cations, which makes it error-prone. You should not use it to generate
an insert for cloning because it is highly likely that the product will
contain at least one mutation. To amplify RNA, use the MasterAmp™ High
Fidelity RT-PCR Kit instead. It uses an enzyme blend with proofreading
activity to obtain a PCR fidelity that is several times higher than Taq.
To amplify a DNA fragment for cloning, the FailSafe™ PCR System
uses a unique enzyme blend and specially optimized PCR PreMixes that
permit amplification of even the most difficult templates with extremely
high specificity and sensitivity, and with a fidelity several-fold better
Tip # 4. Repair your ends.
Double-stranded DNA inserts obtained by PCR often have 3´-non-template-encoded
nucleotides or otherwise imperfect ends for cloning. Special T-vectors
can be used for cloning such PCR products, but we obtain even better
results by blunt-end cloning after repairing the ends of
the insert DNA. T4 DNA polymerase is excellent for this purpose because
it has a 3´-to-5´ exo activity in addition to the polymerase
fill-in activity, so the ends are perfect. This is followed by treatment
with T4 polynucleotide kinase to make sure that all of the 5´-ends
are phosphorylated and clonable. Alternatively, incompatible or damaged
DNA ends can be easily blunt-ended and 5´-phosphorylated in a single
reaction with the End-It™ DNA End-Repair Kit, which
contains both T4 enzymes optimized for this use.
Tip # 5. Increase insert cloning efficiency by eliminating
Treatment of the vector with a DNA phosphatase will prevent self-ligation,
but permit ligation of the 3´-ends of the vector to 5´-phosphorylated
ends of the insert DNA. The nicks on the unligated strands are then repaired
in the host cell following transformation. Epicentre introduced the first
thermolabile DNA phosphatase (HK™ Phosphatase, from an Antarctic
bacterium) in 1988, but it is no longer the best solution for cloning.
Tip # 6. Use a good ligase.
Our Technical Services Scientists have heard about many instances in
which the ligase made the difference between success and failure in
a cloning experiment. Ligases vary in quality. At Epicentre, we go
to great lengths to make sure our ligases are the highest possible
quality, and that makes a difference. We and our customers have had
good success with the Fast-Link™ DNA Ligation Kit, which is optimized
for high-efficiency room-temperature ligations in 5 to 15 minutes,
depending on the type of DNA ends being ligated. Don't try to save
5 more minutes here. Use the right amount of time that is optimal for
the type of end you have. For optimum transformation efficiency, the
DNA ligase in the reaction should be heat inactivated at 70°C for
15 minutes before transforming the competent cells. In some cases,
it is also beneficial to ethanol precipitate the ligated DNA in order
to reduce salt content for electroporation.
Tip # 7. Use competent cells with an optimal genotype
for your cloning purpose.
Competent cells should be chosen based on their transformation efficiency
and their ability to support specific vector features. All of Epicentre's
competent cells retard degradation of plasmid DNA (endA 1 minus);
are defective in recombination, so plasmids are maintained as monomers
(recA 1); allow efficient cloning of methylated and nonmethylated
DNA (restriction minus); and can be used for blue/white screening (lacZΔM15).
For library construction, they also accept large DNA fragments without
size bias up to at least 23 kb, to greater than 145 kb, depending upon
the cells. Please see our competent cells selection guide.
Tip # 8. A kit may be the best option.
Some people think they will save money by assembling all of the individual
reagents themselves. To be honest, it's probably less expensive (and
easier) to buy a kit, such as the CopyControl™ cDNA, Gene & PCR
Cloning Kit. It contains a linearized and dephosphorylated vector,
DNA Fragment Precipitation Solution, end-repair reagents, Fast-Link
Ligation reagents, DNA size markers, competent cells, lysis reagents,
and controls, all of which are optimized and pre-tested. Even more
extensive kits are available for construction of complete BAC, fosmid,
or cosmid libraries. There are also three different kits for screening
recombinants based on insert size, PCR, or restriction analysis.
| ||U ||C ||A ||G || |
|U ||UUU Phe |
|UCU Ser |
|UAU Tyr |
|UGU Cys |
|C ||CUU Leu |
|CCU Pro |
|CAU His |
|CGU Arg |
|A ||AUU Ile |
|ACU Thr |
|AAU Asn |
|AGU Ser |
|G ||GUU Val |
|GCU Ala |
|GAU Asp |
|GGU Gly |
|AUG initiation codon is in bold green. |
GUG codes for Met if in the initiation position.
Stop codons are in red.
|Alanine ||Ala ||A ||89 ||71 ||0 ||0.31 |
|Asparagine ||Asn ||N ||132 ||114 ||0 ||0.60 |
|Asparagine/Aspartic Acid ||Asx ||B ||- ||- ||- ||- |
|Glutamine ||Gln ||Q ||146 ||128 ||0 ||-0.22 |
|Glutamine/Glutamic Acid ||Glx ||Z ||- ||- ||- ||- |
|Histidine ||His ||H ||155 ||137 ||+1 ||0.13 |
|Leucine ||Leu ||L ||131 ||113 ||+1 ||1.70 |
|Methionine ||Met ||M ||149 ||131 ||0 ||1.23 |
|Proline ||Pro ||P ||115 ||97 ||0 ||0.72 |
|Threonine ||Thr ||T ||119 ||101 ||0 ||0.26 |
|Tyrosine ||Tyr ||Y ||181 ||163 ||0 ||0.96 |
* Also add +1 for an N-terminal free
amine and -1 for a C-terminal free carboxyl group.
** Calculate hydrophobicity for a peptide or protein as described
in Fauchere & Pliska. (1983) Eur. J. Med. Chem. 10,