i am doing Phd and cloning few genes from yeast i get partial sequence by standered methods than i did inverse pcr after autoligattion but fail than i did RT PCR by using SMART RACE after isolation Total RNA butr unfrotunatly still not able to get band i follow the methods of manuals and also some my own addition after failing but still no successed. total RNAis oK i get smears always even after nested PCR with touch down etc please help me
you mean sequence of partial gene which i have already and by this i designed primers?
Hi Aslam,
I'm not sure what you did PCR on, DNA or RNA?
I guess, you've tried to PCR a sequence you introduced into yeast, right?
Check if there are restriction sites inside your sequence that might have been digested during your cloning procedure!
Or do you get truncated RNA products from DNA sequences you cloned into yeast? In this case check if your insert has gone in completely.
Maybe you can give as more info,
all the best,
Tom
respected Thomas
let me tell you in detail that i ma doing RT PCR by Smart Race. i have isolated total RNA from my yeast and doing PCR according to the standered manual and given by company long with Smart Race kit too. i have tried my own procedure after little modification i mean chnge in temperature and no.of cycles etc. i have cloned partial sequence of my gene and i designed my two pairs of primer from that sequence. first i did normal PCR and for further amplication i was advised to do nested pcr by using second pair of Primer.but i did not get any band on gel only smears comes usually it doesnt happens when some one use smart race kit but with unfortunalty it happend. now i think it s clear to you. my sequence doesnt have enzyme cut site etc. no i am doing from begning i did not intoduce any sequence yet to the yeast
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