design of PRC primers of full ORF - (Nov/15/2005 )
Hi all, im new here and new to molecular feild ...so please i will be so thankful if you can help me...im designning primers for full ORF for PCR and adding Restriction enzymes termini to both sides....i understood the RE stuff im not sure about the desigh of anti-sense primer (3`). ORF stops with stop codon so should I include this codon into the primer or the one just before it ....??? or stop codon doesnst matter at all in the primer...??? please help to understand this...thank in advance.....have a great day!
Well, I think you want to clone your ORF into an expression vector, don´t you?
So if you want to have apprpriate protein expression it is definitley necessary to include the stop codon. You can include this into the primer sequence or you choose a primer sequence more downstream.
Well, the antisense primer is a sequence complementary and reverse to your basis sequence, e.g.
Your antisense primer might thus be: 5´....TTCGGCTTCGCG...3´
Cheers and good luck!
If you have an IRES sequence (i mean "your protein IRES Antibiotic resistance" or "your protein IRES GFP") you musn't get the stop codon in your amplification...
Kathy, please share more you exp...
thanx everyone you have been so helpfull....but im a little confused....my vector is pGEX-KG:
....Analytical biochemistry volume 192 issue 2, 1 february 1991, pages 262-267, Guan and Dixon
if link is not working please use the science direct to eccess the article ...sorry
and i want to cut my cDNA from another vector (that will not include antibiotic resistance), do PCR to attach restriction termini to it and transer it into the pGEX-KG vector....EcoRI-XbaI site....so if i include the stop codon, trascription will not go beyond my gene...so Amp site will not be expressed.... ...but there is an example you can see down to the pGEX-KG vector...pKG-PTPU323 they have used this vector to express PTP protein and they have introduced a stop codon at pos 323 and still got ampicillin resistance.... what do you think about this? sorry taking too much of your time.....thanx a lot again....
N.B> i am thinking of changing EcoRI-XbaI digest to EcoRI-XhoI because EcoRI and XbaI seem too close to each other