What are the important points to remember when PCR cloning a gene?

Question

I want to PCR clone a gene from mRNA. I know about adding restriction sites to the primers but how far down from the stop codon should I look for a good primer from the mRNA sequence. i.e. what is the polyA tail sequence I should include in my PCR product?

Answer 1

That is one of the problems of going from the mRNA, you will have to design the primer in the translated region meaning you'll have to go inside of the polyA tail sequence. I assume you are isolating your RNA using some type of polyA tail kit.

You can use an oligodT type primer instead of a specific one, and just use a 5' specific primer, but a lot of times that creates problems in the cloning part of your experiment. If you are looking to put it in an expression vector why go from mRNA, why not PCR clone from genomic?

Answer 2

is this why I have a tough time making new friends? I read Just Me's answer. I do all of those things immediately...I need to know from the start if I want to waste my time on this person or not..you can find out a lot in the first five minutes. have you ever had an abortion? do you whore it up? are those real? say one thing bad about the Sox and I'll cut you are you a republican or a democrat? :| do you litter? etc etc

Answer 3

You need to heat the strand to separte it...sry if that doesnt help