how to isolate gene through PCR - (May/21/2008 )

Hi,
I want to isolate sugar transporter gene from plant to overexpress it through constitutive promoter.the length of the gene is 1685 bp long. i want to isolate total gene including everything like orf n others things to express it as natural gene.please help me how to proceed forward.
Thank you very much.
SVRREDDY.
Ph.D student.

Hi,

it sounds like you have isolated the coding region of your gene but you are interested in also finding the 5' and 3' UTR as well as the promoter sequence.

My suggestion is to use 5' and 3' RACE (a kit would be good) and design primers starting from your known cDNA sequence to find your 3' and 5' UTR (respectively). To find the promoter, you will then genome walk starting from the known sequence that you have isolated in the 5' UTR into the promoter region.

Keep in mind that plant promoters can be really really long so if you have some clue as to promoter sequences or other features that you may be looking for that could be very very helpful.

If you just need the coding region to clone in an expression cassette, just design primers for both ends of the gene...

You can insert a few bp ahead of the starting codon just to make sure you have the ribosome binding site that is specific for your gene. Other than that, check if you need to insert a stop codon or not. It depends on whether you'll fuse this protein with something else.

And if you are fusing this protein with something else, make sure they are in frame.

[quote name='genejockey' date='May 21 2008, 08:17 AM' post='136676']
Hi,genejockey',
i want to express this gene under the control of Hsp promoter in vector.i want to see the expression of this under high temperature for that which part of gene i needed to clone into vector contaning hsp promoter. how can i desigh primers to isolate that part. please help me in this issue and thank you very much for your early reply.
SVRREDDY