3 replies to this topic

[lab_member]

I am just curious whether a ligation mix can be used after storage for 1 month in -20'C (freezer).

One of my lab colleagues used the ligation mix for transformation then screened for colonies by colony PCR. The result was that there was no inserts. She actually used the same ligation mix when it was 'fresh' and got quite a lot of inserts.

Do you think that the DNA has already degraded even i  -20'C?

[Nrelo]

lab_member, on Mar 26 2009, 11:55 PM, said:

I am just curious whether a ligation mix can be used after storage for 1 month in -20'C (freezer).

One of my lab colleagues used the ligation mix for transformation then screened for colonies by colony PCR. The result was that there was no inserts. She actually used the same ligation mix when it was 'fresh' and got quite a lot of inserts.

Do you think that the DNA has already degraded even i  -20'C?

i think the problem is due to the polymerase rather than DNA. DNA itself is quite stable in-20C. But the polymerase is only stable in its storage buffer when it is in -20C

[lab_member]

Nrelo, on Mar 27 2009, 04:02 PM, said:

i think the problem is due to the polymerase rather than DNA. DNA itself is quite stable in-20C. But the polymerase is only stable in its storage buffer when it is in -20C

Actually the polymerase she used is stored in storage buffer. The ligation mix is of the plasmids and the ligase... I don't think it is the problem of the polymerase as there were primer dimers...so I don't what what went wrong...

[Nrelo]

I misread you post . but anyway, I once stored my ligation mix in 4C for 2 weeks and it worked fine

Edited by Nrelo, 29 March 2009 - 02:09 AM.