Insert seems to stimulate self ligation of pGEX vector using EcoRI single digest method

Hans Wessels hansw at sci.kun.nl
Sun Aug 25 06:19:44 EST 2002


Hi David,

Thanks for your effort to help me out. I have also considered some of
the possibilities you mentioned and i'll explain it to you.

The cleanup kit used to purify the pcr product does not purify
fragments below 250 bp. So if there were primer dimers they couldn't
acount for the strange results. Same thing for the digested vector;
the sequence that has been cut couldn't be ligated back into the
vector since it was not purified by the kit (about 10 bp  fragment).

PCR: my pcr reaction uses official primers for the screening on
insert; when i screen a cfu with an empty vector i get a ~100bp
product (MCS and linker sequence to GST fusion protein) so it is
possible to distinguish the difference between a not working reaction
or an empty vector. I screen one CFU in each reaction. I also do a
screening pcr with the forward vector primer in combination with the
reverse gene primer; if the gene is in the right orientation i get a
~1,3kb pcr fragment, if not or if it's not there i do not get a pcr
product since either the primers point into the same direction or the
reverse gene primer cannot anneal because there's no gene. In both
reactions a negative controle is tested to insure proper results.
Considering the out competing possibility you mention; i believe it's
not: let's say we have about 50% pGEX 4T1 and 50% Syn I pGEX 4T1 in a
CFU; i get a proper 100bp and a 1,4 kb product; i haven't seen this
happen in this way but i have done a pcr screening on BOTH a negative
controle (empty vector) and a positive controle in one screening
reaction yielding a 100bp product AND a 1,4kb pcr fragment.

Considering the inactivation step: i have noticed that it doesn't
always make a difference... Some genes (i've been working on 5 of them
in this research) in combination with the used pGEX 4T1 vector gave a
very low transformation efficiency. It was able to raise efficiency
using inactivation with about 20%. It is possible that when you use
ultra-competent cells in combination with a very efficient ligation
you do not notice the difference since this yields very big amounts of
CFU, but in my case it was worth the effort.

Thank you for your time,

yours sincerely,

hans



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