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Cloning before sequencing- is it necessary??? - (Sep/13/2010 )

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There is difficulty at both ends of a sequence run: the 5'-end has problems for a short period, and the 3'-base calls get progressively weaker as the sequence extends. The solution is to sequence each clone in both directions, preferably using primers that bind to the vector about 30 bp to either side of site into which your fragments are cloned. With ~400 bp fragments, you'll get complete coverage...

-HomeBrew-

Thank you all for the advice.

Since, I am basically looking to screen SNPs, I am thinking of sequencing the PCR product directly. It will be quicker and will also give me what I require.

Now to do that, do I need a different primer for sequencing purposes??? or can my PCR primers do the job?

-gt_ameya-

Sequencing read length depends mainly on the instruments and chemicals used. For example ABI instruments can have capillaries of various length, various types od polymer inside, so you may get up to 700-800 bp reads or only around 300-450. In shorter reads less bp ale lost on the beginning, also ABI v1.1 kit is better than v3.1 if you want to read closer to the primer.

Once you know how long reads you will get and what type of sequencing kit you are gonna use, then you can design your primers. For example if you know you will get good 450 bp sequence, starting 40 bp from the primer, then you design forward primer 45 bp upstream of beginning of the exon and make maximum 900 bp amplicon to sequence using only PCR primers. If you have longer amplicon, then use inner sequencing primers. You can derive your amplicon from DNA having primers in introns, if exons have suitable length and numbers (5 exons is not much, so I would recoment this, one amplicon on each exon). Other way is to transcribe RNA to cDNA and amplify the whole coding region, this is practical if there are lots of small exons and overal length of coding region is up to say 1kb (and if you have the RNA of course). So this also depends on your gene.

-Trof-

gt_ameya on Thu Sep 16 08:51:25 2010 said:


Now to do that, do I need a different primer for sequencing purposes??? or can my PCR primers do the job?


you can, but it is not recommended. sequencing primers may be longer than pcr primers (~30 bases). they should be cleaner. you don't want to start from the very end of the fragment to avoid slippage.

-mdfenko-
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