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Sequencing


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#1 Lotus Japonicus

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Posted 17 July 2009 - 09:09 AM

I'm sorry if this is terribly basic but I thought it best to ask as soon as possible. I have a 2.8kb gene which I wish to sequence, and no quidance on how to do this (everyone on holiday) thought it's pressing. I presume that I need to amplify my gene and then sequence from that amplification? Can I, say, amplify a 1kb fragment and then do several 300bp sequencing reactions of the same fragment (say, with one primer at 10-30bp, one at 280-300 and one at 580-600)? I'm having a little trouble designing primers in some of the regions so that would definetley simplify things.

Thanks a lot for the help.

((p.s. amplifying from gDNA))

#2 mdfenko

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Posted 17 July 2009 - 11:20 AM

what method are you going to use to sequence? are you going to do manual sequencing (gel and all)?

machine sequencers can get 700-800 bases (depending on reaction strength and capillary length and running program...) with good accuracy. but you can't read from immediately after the primer.

if you make 1000 base, overlapping fragments then you can use forward and reverse primers to get the entire fragment sequences then assemble your gene.
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#3 Rsm

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Posted 17 July 2009 - 07:36 PM

I agree with mdfenko, you can sequence your whole 1kb PCR product using the same primer that you used for amplification. You will lose some nt at the ends, however.
If you wish to sequence your whole 2.8kb gene, design a primer for every 500nt forward and one reverse at around +300bp (ie. positions +1, +500, +1000, +1500, +2000, +2500 and -300). You can use an alignment program like Codon Code Aligner (http://www.codoncode.com/), whose demo version is free of charge and really good.
Cheers,
Minna
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