Insert seems to stimulate self ligation of pGEX vector using EcoRI single digest method
hansw at sci.kun.nl
Sat Aug 24 08:05:42 EST 2002
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:
Ligation composition: vector,T4 DNA ligase,buffer,insert
Negative controle: vector,T4 DNA ligase,buffer,MQ (instead of insert)
Total volume of either ligation: 10 microliters
Incubation: 2hrs at room temperature
!DURING LIGATION I USED A MASTERMIX CONTAINING VECTOR,T4 DNA LIGASE
AND BUFFER FOR BOTH THE LIGATION AND THE NEGATIVE CONTROLE! This way i
ensured that both the ligation and the neg. controle were exactly the
same (besides the insert ofcourse).
Next, i inactivated the T4 DNA ligase by incubating for 20 minutes at
65 degrees celcius.
Transformation was carried out as follows:
200 microliters of competent E. coli XLI Blue cells were pooled and
100 microliters was transformed with 5 microliters of the inactivated
ligation solution and the other 100 microliters were transformed with
5 microliters of the inactivated neg controle ligation solution.
Transformation was carried out using the heat-shock method. Both the
ligation and the negative controle were plated on LB-plate with
ampiciline and glucose in exactly the same quantities. After O/N
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.
Considering the CFU ratio of ligation:negative controle (13:1) you
would expect a succesfull ligation yet it seemed that none of the CFU
found contained the insert but did contain the vector. The experiment
was repeated and yielded the same results. When we conducted this
experiment with an other gene using the double digest (BamHI/EcoRI)
method it resulted in a succesful ligation/transformation.
Is it possible that the insert stimulates self-ligation of the vector
using this single digest method? I hope you can help me out because
i'm a bit lost explaining this result.
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