pGEM vectors - (Nov/05/2007 )
Hello All,
I am trying to clone a 7kb fragment in TA clone and its not working. I wanted to give a try to pGEM vectors, if that could work but there is a tiny problem.
When we received the kit, the vector wasn't aliquoted and it was thawed and frozen at least 10 times so far. It seems that this cloning doesn't work anymore. I checked the manual quickly and couldn't find anywhere that such vector should be aliquoted but couple of guys in the lab stress that it should be aliquoted otherwise the enzyme sitting on the end gets distroyed and it doesn't work.
I would like to know from regular users if this is true. I checked the manual quickly (running out of time for the deadline) but didn't find any of such stuff. The questions are:
1. If this aliquoting and terminal enzyme thing is true?
2. If not, what could be the reason that such cloning stopped working.. (The kit was stored properly in -20C)
3. If the guess is that the T-overhangs are gone, is there anyway to put them back (like we put A-overhang on the PCR product?)
4. If the rapid ligation buffer is messed up (loss of dATPs), can I use regular ligation buffer that we get with T4 ligase?
Thanks a million in advance.
1. If this aliquoting and terminal enzyme thing is true?
2. If not, what could be the reason that such cloning stopped working.. (The kit was stored properly in -20C)
3. If the guess is that the T-overhangs are gone, is there anyway to put them back (like we put A-overhang on the PCR product?)
4. If the rapid ligation buffer is messed up (loss of dATPs), can I use regular ligation buffer that we get with T4 ligase?
We usually use pGEM-T Easy vector system from promega and they advice in the manual that the vector should be aliquoted all in order to avoid repeated freeze-thaw. We also aliquote rapid ligation buffer that comes with the kit (single use).
You can put A-overhang back in the product. Into the vector... I never tried but there is another topic here somewhere that explains something like that.
I don't know if you can use other ligation buffer. What you should do is always vortex the vial with rapid ligation buffer before each use (as I said we aliquot single use, so we don't have to do that).
Good luck!
Hi,
Have you tried T/A cloning using different PCR product that's smaller in size than say 7 kb?
Also, since your PCR product is 7 kb (I'm guessing that you wanted to minimize random mutation?), so... there's no by any chance that you're using a proof reading DNA polymerase? If you're using a mixture with Taq, forget about this.
Who was the last one using the kit before you and how did the ligation and cloning work for that person?
>If the guess is that the T-overhangs are gone, is there anyway to put them back (like we put A-overhang on the PCR product?)
---> Incubate pGEM-T vector with 1X PCR buffer, 2 mM dTTP with Taq (1U/ug plasmid DNA/20 ul rxn) at 70 to 72C, 2 hrs. Sourced from Marchuk et al 1991.
Reference:
Marchuk et al. 1991. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR product. Nucl. Acids Res. 19(5): 1154 (Free fulltext in Pubmed).
...-...
Have you tried T/A cloning using different PCR product that's smaller in size than say 7 kb?
I suppose you mean to say using this kit, if so, No I haven't but the one who used before me claimed that it doesn't work as he hasn't aliquoted the vector.
Also, since your PCR product is 7 kb (I'm guessing that you wanted to minimize random mutation?), so... there's no by any chance that you're using a proof reading DNA polymerase? If you're using a mixture with Taq, forget about this.
Sorry, I didn't understand what you mean here. I used Qiagen's long-range PCR kit and it has proof-reading polymerase but at the same time it has terminal transferase as well, to add A-overhang. Now, I didn't understand what you said about Taq (yeah, sure amplifying 7kb with only Taq and expecting no mutation is a fool's job)
If the guess is that the T-overhangs are gone, is there anyway to put them back (like we put A-overhang on the PCR product?)
---> Incubate pGEM-T vector with 1X PCR buffer, 2 mM dTTP with Taq (1U/ug plasmid DNA/20 ul rxn) at 70 to 72C, 2 hrs. Sourced from Marchuk et al 1991.
Reference:
Marchuk et al. 1991. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR product. Nucl. Acids Res. 19(5): 1154 (Free fulltext in Pubmed).
You are a life-saver, I will give a try and if I succeed, I will make sure to leave a note here.
Thanks a million