Stuck from last august! need help urgently - cloning and pcr !
Posted 05 February 2013 - 05:07 AM
I have been trying to clone a pcr product of 3065bp in pEF6V5 His TOPO vector. I have tried everything but couldnt amplify my product with normal taq. I have tried phusion (neb), recombinant taq (fermentas, sigma, invitrogen, intron biotech), dreamtaq (fermentas), platinum taq (invitrogen) and also Ex taq from takara but in vain as none gave any result. Thought phusion (neb) gives band of same size as required but it can not be cloned in TOPO due to 3'A missing.
based on many TOPO related forums i tried to standardize ploy A addition after amplifying my product with phusion (neb) but in vain!
What i am looking for is some taq polymerase or some way by which i can amplify my pcr product with any normal taq.
My primers are Fwd: 5’ ACATGCTTTGGGACTGCCACTGA 3’
Rev: 5’ GCCGGCAATGGACGTGAACA 3’
I am using 1x buffer, 2.5mM mgcl2, 1.25ul (0.5 uM) primers, 0.25mM dntp, 1U/ul Taq, 1ul (<500ng) template (cDNA) for a 25ul reaction.
The pcr conditions are
94 - 4 mins
94 - 45 secs
X - 30
72 - Y mins
72 - 10 mins
4 - hold
I have tried gradient of X from 55 to 68 but no result. And Y from 2 to 4 minutes but no result.
Posted 05 February 2013 - 07:39 AM
Posted 05 February 2013 - 08:14 PM
I have tried adding 3'A overhang but this doesnt work for me.. I am planning to add restriction sites at 5' end of my primer. There is a unique BstXI site in my vector. Is it okay if i use this topo vector for restriction based cloning instead of TA cloning.. i am worried if topoisomerase wud interfere with the latter type!
Posted 06 February 2013 - 12:44 AM
Posted 06 February 2013 - 02:33 AM
Have you try adding 5% DMSO ? I usually add 1.25 uL of DMSO into 25 uL of PCR reaction.
This will help to stablize DNA template.
also "1ul (<500ng) template" might be too much to start with ?
I would do a ten fold less DNA template or even 1/100 dilution.
hope this have helped
Posted 06 February 2013 - 06:18 AM
I strongly agree with Lisa that you should dilute your template DNA. You might also try adding 3-8% betaine 1M solution (Qiagen "Q-solution") instead of the DMSO.
Is your template high GC?
Posted 06 February 2013 - 08:06 AM
Posted 06 February 2013 - 11:33 AM