APOE PCR conditions - RFLP e2, e3, e4? (Mar/07/2003 )
I am trying to genotype the different alleles of APOE: e2, e3, e4.
Does anyone have a good PCR that will amplify the region?
I am having a lot of problems with the gene...
some days ago i amplified the ApoeE gene by RT-PCR, the base pair is 900 ,
and the condition is
the first PCR product have two bands, and then i use the product as a template of the second PCR. the condition is same as above.them i get the ApoE gene. i sent it to sequence. the sequece is right.
wish you goog luck.
Thank you for your reply, Greenbee.. actually: I got it to work!
The magic ingredient is 2.5% of formamide (I had already tried, DMSO, Tween, ammoniumsulfate, MgCl gradient, touchdown pcr, annealingtemp optimizations, other techniques.. all of which didn't work). The primers I used are from Zivelin et al. a 220 bp product.
now ,i have a problem in expression of apoE.i insert the apoE gene into the vector of pGEX-2T to express the fusion protein.but it doesn't work! Then i put it into the vector of pQE30,it doesn't work neither.what can i do? is the 900 base pair gene dosen't express in procaryotic cell ?
can you help me? give me some advice.
Sorry, I don't have any experience w/ vectors.