insering cDNA before the tag.... - what problems should I consider? (Jun/11/2006 )
i want to insert cDNA (PCR product) before the tag....so I should desing primers so that there is no stop codon of cDNA and cDNA and tag should be in frame....what other problems should I consider? will my protein be expressed without the stop codon? thank you very much?
so i made reverse primer to end just before the stop codon.....but it is so bad... ...it has potential for hairpin and self annealing structures....how far before stop codon can I cut? can I manipulate a little or its just what i have? thanx a lot for any help.
Yes, your protein will be expressed together with your tag. However, if your protein must be expressed in a specific membrane (organelle membrane, plasma membrane), the tag may interfere in this expression make it difficult.
You can cut as far as you want as long as the native properties of your protein you want to study don't change. Nevertheless, the closer to the stop codon you obtain your protein, the closer to the native protein and the reality you will be.
Just make sure the cDNA and tag are in frame, usually that where it problem lies. Also if the tag is in the 5', then there might be the problem of translocation or if its a secreted protein, then the possibility of cleavage.
you may have to insert few aminoacid sequences between tag and yourprotein if the last aminoacid of the tag holds strong differences in chemical properties.
After that matter, you may also consider the possibilty of cloning it after the tag sequence too.
check out the Roche website. They have a good manual for starter. You have to download the manual. I am just providing the link for introduction
thank you so much for the suggestions! what about the reverse primer ive desinged? i just order it in spite of the fact that it can form hairpin and self annealing structures? i think there is no other way right? maybe inserting like Fred suggested.....
the two possibilities are ok.
I'ev inserted Flag sequence by restriction enzymes experiment, but i've also done succesful PCR with a Flag tail without many and many adjustments of PCR conditions.
Using DMSO helps.
thank you very much, Fred ill try PCR and hope it will be ok...anyway im cutting out of the gel, that is my preferred method...so if there are some dimers or other structures it wont matter much right?
you can first try to run an aliquot of your native sample, and an aliquot of sample pheated 5' &t 60°.
I've seen when i tried amplifying a sequence containing a siRNA sequence, that resolution was accurater with heating. (removing strong secondary structure)