Insert seems to stimulate self ligation of pGEX vector using EcoRI single digest method
dmicklem at cmgm.nospam.invalid
Sat Aug 24 11:57:16 EST 2002
In article <9bb75204.0208240505.7582df6a at posting.google.com>, Hans
Wessels <hansw at sci.kun.nl> wrote:
>I have encountered a problem i cannot quite comprehend. I have tried
>to ligate a gene into the pGEX 4T1 vector by means of single digest
>with EcoRI. I will try to explain the method and the resulting problem
>so that you can try to help me out.
>The general idea is as follows:
>PCR on gene (template: chrom. DNA) with specific primers. Both forward
>and reverse primers contain an EcoRI restriction site. This results in
>an ~1300 bp pcr product with EcoRI restriction sites on both sides of
>the gene. After purification of the PCR product it is digested with
>EcoRI and again purified using the QIAEX II DNA purification kit.
>After each step the pcr product was checked by means of agarose gel
>Next the pGEX 4T1 expression vector was digested with EcoRI and
>purified with DNA purification kit (checked on agarose gel electr.).
>Then the vector was treated with CIAP (to reduce self-ligation of
>vector during ligation) and purified again (again checked on agarose
>gel electr.). In short; neither the digested vector or insert solution
>contained any restriction enzymes left from earlier stages of the
>method. Ligation was carried out as follows:
>Next, i inactivated the T4 DNA ligase by incubating for 20 minutes at
>65 degrees celcius.
>incubation at 37 degrees celcius we counted the CFU. As you might
>expect the ligation plate had 13 fold more CFU than the negative
>controle. After screening of 60 CFU's by doing a PCR on the CFU's
>using vector primers it showed that NONE of the 60 CFU screened
>contained the gene but DID CONTAIN the vector. Recall that everywhere
>along this experiment everything was done using mastermixes; so
>differences in the ligation and transformation solutions/cells cannot
>acount for the result.
Did you gel purify your original PCR product? If not, could you have
cloned contaminating primer-dimers? Even a very small mass of primer
dimer (ie hard to see on a gel) could contain a large molar excess over
your 1.3kb product. Can your PCR screen distinguish completely empty
vector from a very small insert?
Another (slim!) possibility: could contaminating empty vector in your
PCR screening step be out-competing your correct clone? This can
obvously be excluded if you had a negative control (no template added)
for your PCR....
FWIW, I never bother heat inactivating ligase before transforming. Do
you find this step makes a significant difference?
D.R. Micklem, Time flies like an arrow...
Dept. of Genetics, Fruit flies like a banana.
Cambridge University, Email:drm21 at mole dot bio dot cam dot ac dot uk.
Cambridge, UK Phone: +44 (1223) 766336
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