cloning PCR products

tfitzwater at gilead.com tfitzwater at gilead.com
Mon Jan 15 16:26:58 EST 2001


>Yin Wang (yin.wang at weizmann.ac.il)
>Sun 14 Jan 2001 - 16:16:10 GMT
>Hi all,
>I'm looking for an efficient way to clone 1.6 and 2 kb PCR products into
>pET 20b(+) vector,after trying in vain with cohesive end[EcoR1 ,Hind111]
>ligation for months.
>Thanks in advance!!

1.  Did you include a GC-rich clamp upstream of the restriction sites when
you designed your primers?  We place ccg upstream of the Hind II site and
gcc upstream of the EcoR I site.

2.  Exonuclease contamination becomes more of a problem in cloning when
sequential digestions are performed.  In my hands, the number of inserts
increased when a simultaneous digestion was performed.  Promega and
Fermentas are more strict about the levels of exonuclease they allow in
their products than most suppliers, and these have performed better in my
hands.  One prominent supplier has used white-out on their spec sheets when
I specifically requested that the exonuclease levels conform to their
catalog specs (they do not always conform).

3.  Are you using enough enzyme?  The reaction volume must be at least
10-fold greater than the total volume of restriction enzymes in order to
maintain a low glycerol concentration.  Excess glycerol can lead to star
activity.  Incubate at 37°C for 3 hours (80 units of each restriction
enzyme x 3 hours = 240 unit hours).  In my hands, I can cut clean DNA to
completion in 3 hours using 80 units each of EcoR I and Hind III with 14.4
pmoles (0.66 µg) of a 1000 bp PCR product (the insert).  On the other hand,
excess enzyme will increase the probability of exonuclease problems.
It also takes 240 unit hours to cut 14.4 pmoles (25 µg) of vector.  When
preparing my own vector, such as pUC19, I include 3 units of shrimp
alkaline phosphatase in the digestion so that the vector is simultaneously
dephosphorylated.  By combining the restriction with the dephosphorylation
step, you save time and you don't lose any DNA during an intermediate
clean-up step.
Units are generally defined as the amount of enzyme required to cut 1 µg of
a particular substrate DNA in 60 minutes at the appropriate temperature in
a 50 µL reaction.  Unit hours are a way of thinking about the restriction
of a given number of pmoles or molecules of DNA that you don't get with
micrograms.  25 µg of vector and 0.66 µg of a 1000 bp PCR product are not
intuitively equivalent, but 14.4 pmoles (3.5 x 10e11 molecules) of DNA
containing a single copy of each restriction recognition site will cut in
the same amount of time with the same number of units, provided that there
is not a significant difference when linear and supercoiled DNAs are
compared.
Uncut PCR product should travel slightly slower than the cut material.  If
a faint band exists parallel to the uncut control, then repeat the
restriction cut for 60 minutes.  If the analytical single-cuts indicate
that only one enzyme failed, then repeat the cut with that enzyme only.  It
is normal for the cut PCR product to appear as two bands (presumably
single- and double-cut DNA fragments?), both traveling faster than the
uncut material.  There will be sufficient double-cut PCR product to
proceed.  The vector, however, should be cut to completion.  ANY PCR
product traveling below the expected band (primer dimer?) will completely
overwhelm the cloning  reaction.  If primer dimer exists, then a
preparative gel must be run to eliminate primer dimer from the ligation
reaction.  Failure to gel purify may result in selective (> 95%) ligation
of primer dimer into the vector.  If primer dimer is completely absent,
then the material may be Tris-buffered phenol:chloroform extracted,
chloroform extracted and ethanol precipitated with ammonium acetate.  If
purified in a Centrex 30K cartridge instead, restriction enzymes (and SAP)
will remain and interfere with ligation.  Clean DNA is critical for optimal
cloning.

4.  For the ligation step, a 2:1 (1.0 pmoles:0.5 pmoles) insert:vector is
recommended for this range of insert size.  If the vector was not
gel-purified, follow the ligation reaction with a heat-inactivation step
and restriction of another site in the multiple cloning site that is absent
in the insert as a quick way to eliminate religation of any single-cut
vector.

Good luck,

Tim Fitzwater
Principal Research Associate
Gilead Sciences


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