primer conditions for expression work - (Jun/12/2008 )
Hi all,
Now that I have understood about how to design primer from ORF region for expression work, thanks to all of you, I have one more question in my mind.
since these primer contain RE sites and Kozak and they are designed from start codon and stop codon I dont have much freedom to mentain primerdimer, GC content and self dimer conditions. Can they still work. what should be the ideal PCR condition. What DNA polymerase should I use?
thanks in advance once again.
regards
Vani
since these primer contain RE sites and Kozak and they are designed from start codon and stop codon I dont have much freedom to mentain primerdimer, GC content and self dimer conditions.
Correct, you don't.
The best thing you can do is design primers with a high Tm. I design the Tm of my primers around 70C - that's 70C for the section of the primer that is complementary to your template (not the whole primer). This will allow you to use a high annealing temperature, which will help prevent the primer dimers and so on from forming and increase the specificity of the primers for the template.
Phusion polymerase from Finnzymes. Search the other posts for why this is the best cloning enzyme. I've written about this a couple of times.
Good luck, Rob
since these primer contain RE sites and Kozak and they are designed from start codon and stop codon I dont have much freedom to mentain primerdimer, GC content and self dimer conditions.
Correct, you don't.
The best thing you can do is design primers with a high Tm. I design the Tm of my primers around 70C - that's 70C for the section of the primer that is complementary to your template (not the whole primer). This will allow you to use a high annealing temperature, which will help prevent the primer dimers and so on from forming and increase the specificity of the primers for the template.
Phusion polymerase from Finnzymes. Search the other posts for why this is the best cloning enzyme. I've written about this a couple of times.
Good luck, Rob
Thanks Rob,
How about using taq, as our lab has ample of taq supply.
Just a thought:
If you are making an expression contruct by PCR, have you checked if your expression clone is already avaialble from big resources? Even full length cDNA clone may be good to have, if for nothing else, for use as a template in PCR.
..
If you are making an expression contruct by PCR, have you checked if your expression clone is already avaialble from big resources? Even full length cDNA clone may be good to have, if for nothing else, for use as a template in PCR.
..
I know that would be lot more easier and less work to do , but I dont have it available .I checked it on Addgene. no luck. though they have submited the full length sequence in NCBI. What do you mean by bg resources ,can I check it some where else???
Open Biosystems and Origene are other suppliers. With regards to Taq, well, you can use it but your chances of a mutation are much greater. You may get lucky and you'll be fine, more times than not you will, but if you can avoid the problem then why not?