Inclusion of restriction sites ...ERRATUM

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Sun Nov 3 18:58:23 EST 1996


On Sun, 03 Nov 1996 13:25:29 +0000, Andrei Popov <andrei.popov at bbsrc.ac.uk> wrote:
>I cloned many a PCR fragment using this approach, and recently also
>5'RACE products where the percentage of correct clones was exceptionally 
>high.

[deleted]

>
>I find the tone of Dr. Aiyar's comments simply unprofessional and rude.

Unprofessional or otherwise, I have tried this cloning approach
a few years ago and failed on every attempt.  Therefore I did the
following experiment to test what types of ends Klenow left in
my hands.

My standard Klenow conditions are 20oC for 20 minutes, 50 micromolar
final nucleotide concentration.

The plasmid being cloned into was pCDNA3.  My intention was to 
clone my PCR products into the EcoRI site of this plasmid.  The 
relevant portion of the polylinker is shown below.

--gaattctgcagatatcccatcacactgg--
--cttaagacgtctatagggtagtgtgacc--

To test the ends I cut the plasmid with EcoRV then treated with 
shrimp alkaline phosphatase.

The cut plasmid should look like:

5' atcccatcacactgg-----gaattctgcagat 3'
3' tagggtagtgtgacc-----cttaagacgtcta 5'

The number of 5' ends was estimated by a kinase reaction to be 
about 2.2 picomoles per 5 micrograms of cut DNA.

The following treatments were then performed and the amount of
label incorporated was estimated by label retension on DE-81 paper.

The Klenow used was from NEB, 2 units of Klenow were used per
reaction, with 4 micrograms of cut, phosphatased DNA.

a) Klenow + TTP for 20 minutes = less than 0.05 pmoles

b) Klenow + cold dATP, wait 10 minutes, add labelled TTP,
   wait another 10 minutes
   = less than 0.05 pmoles

c) Klenow + cold dGTP + cold dATP, wait 10 minutes, add 
   labelled TTP, wait another ten minutes
   = ~ 5 pmole of label incorporated

d) Klenow + labelled dGTP = ~ 1 picomole incorporated

There are several possible explanations.  The one I favor is
that Klenow left ends that were considerably more ragged than
predicted .... which certainly explains the complete lack of 
clones in my cloning.  At the time we used cells made competent 
by treatment with CaCl2, and my recollection is that we obtained 
transformation efficiencies in the range of 1x10E6 colonies per 
microgram of DNA.

If you are able to clone with this approach, my suspicion is
that either you have used a significantly different ratio of 
Klenow units/ends of DNA, or that your transformation efficiency
allows you to detect an extremely rare ligation event.

Later,
Ashok

P.S. The PCR product I was attempting to clone, did clone when
     self-ligated (after 5' phosphorylation) followed by digestion
     with EcoRI.




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