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APOE PCR conditions


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5 replies to this topic

#1 Niqqie

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Posted 07 March 2003 - 01:59 PM

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... :(

#2 Niqqie

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Posted 18 March 2003 - 11:17 AM

noone?

#3 greenbee

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Posted 24 March 2003 - 07:18 PM

some days ago i amplified the ApoeE gene by RT-PCR, the base pair is 900 ,
and the condition is
94 30s
58 30s
72 60s
36 cycles
72 300s

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.

#4 Niqqie

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Posted 25 March 2003 - 06:29 AM

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.

#5 greenbee

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Posted 01 April 2003 - 04:41 AM

congratulations!

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.

#6 Niqqie

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Posted 01 April 2003 - 09:10 AM

Sorry, I don't have any experience w/ vectors.




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