4 replies to this topic

[alpha]

Hi all..
I am going to do cloning with pGEX-2T vector. My insert is only 180 bp. I got good PCR product and I purified it by gel extraction kit…..its work well… my band is very clear in 2% Agarose gel at correct size.
But the problem when I digest my insert with BamH1 it disappear (No band)…
My restriction enzyme is ok…..No contamination..
No restriction site corresponding to Bam H1 in my insert.
I repeat it .. but still same funny result.
I observed the DNA dose not migrate throw the gel, just aggregate in the Agarose hole.
CAN YOU HELP ME PLEASE..

[alpha]

1240 views and no reply!!!!
Can you help me please...please...please..

[lab rat]

alpha, on Aug 7 2009, 07:58 PM, said:

Hi all..
I am going to do cloning with pGEX-2T vector. My insert is only 180 bp. I got good PCR product and I purified it by gel extraction kit…..its work well… my band is very clear in 2% Agarose gel at correct size.
But the problem when I digest my insert with BamH1 it disappear (No band)…
My restriction enzyme is ok…..No contamination..
No restriction site corresponding to Bam H1 in my insert.
I repeat it .. but still same funny result.
I observed the DNA dose not migrate throw the gel, just aggregate in the Agarose hole.
CAN YOU HELP ME PLEASE..

Hi alpha,

Is there a special reason you want to cut your vector with BamHI?

regards,
lab rat

[cellcounter]

alpha, on Aug 10 2009, 02:07 PM, said:

1240 views and no reply!!!!
Can you help me please...please...please..

In a case like this, so many things have to be considered, your technical steps have to be evaluated alongwith images, controls, reagents, etc  that any reply is sure to be inadequate. So, I guess people just don't venture it, at least I try not to get into writing a long reply.

Best thing is to critically discuss the issue with some experienced colleague in or out of the lab.

[HomeBrew]

What happens when you cut your vector with Bam?  If it looks alright, that rules out a lot of potential problems.  If it acts wierdly too, it's not your PCR prep, but something about the digestion (the enzyme, the 10x buffer, the water, your tubes, etc.).