Left polymerase above fridge for more than 1 week - still working? (Mar/06/2011 )
Somebody left my T4 polymerase, T7 polymerase and T4 ligase (all I bought from NEB, cost me almost USD300) on top of the refrigerator for more than 1 week.
Any chances it is still functioning? any way to assay the efficiency?
Thanks.
Hey Adrian,
Sorry to hear that. I can assure you, it wasn't me 
.
You could do a routine PCR with primers to see if it is still working. Some housekeeping gene amplification. or some internal controls. or some plasmid before the insert.
This comes to me as I am writing to you.
1. Any plasmid you might might have inserted an antibiotic gene cassette. PCR to check if the polymerase is fine
2. Digest.
3. Use Ligase, put in the cassette.
4. Confirm with PCR again...
5. Advantage: You have your own plate of some gene knock out + you know everything is working... 
BTW, why weren't you in the lab for a week?
Hey gt_ameya,
LoLx... I know it wasn't you... 
Actually the T4 and T7 polymerase is mean for generation of LIC fragments, for my own private project. I will take your advice and try it anyway, I was thought they might be a short cut way so that I can avoid doing extra work..
I was in the lab the whole week, but I did not touch or look into the fridge as I'm doing something else, plus, the lab is very messy...
gt_ameya on Mon Mar 7 06:45:47 2011 said:
Hey Adrian,
Sorry to hear that. I can assure you, it wasn't me
. You could do a routine PCR with primers to see if it is still working. Some housekeeping gene amplification. or some internal controls. or some plasmid before the insert.
This comes to me as I am writing to you.
1. Any plasmid you might might have inserted an antibiotic gene cassette. PCR to check if the polymerase is fine
2. Digest.
3. Use Ligase, put in the cassette.
4. Confirm with PCR again...
5. Advantage: You have your own plate of some gene knock out + you know everything is working...
BTW, why weren't you in the lab for a week?
And check the T4 ligase as well. This enzyme denatures easily.