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herrroaa 06-27-2011 05:12 AM

ion torrent
 
Hi all,
I want to pre-amplify a 70Kb gene prior resequencing via ion torrnet. could any one please tell me what is the best way to do this.
Thanks

krobison 07-02-2011 07:44 PM

When you say pre-amplify, from how many samples (and if more than one, how many runs do you want to pack it into)? Do you care only about exons or the entire gene?

herrroaa 07-04-2011 12:09 AM

Quote:

Originally Posted by krobison (Post 45522)
When you say pre-amplify, from how many samples (and if more than one, how many runs do you want to pack it into)? Do you care only about exons or the entire gene?

I want to do this in about 50 samples and i am interested in entire gene.
I think I can do it in one run using the 318 chip.

krobison 07-05-2011 12:20 PM

One possibility would be to design (and validate!) a series of long PCR amplicons to tile the gene (each one overlaps the previous one by a bit).

The pooled amplicons from each sample then generates a new fragment library. One thing to watch out for is that primer-derived sequences will have artificially high variant rates due to primer synthesis errors.

herrroaa 07-06-2011 06:01 AM

There is a long lange pcr kit offered by Qiagen. this kit will generate a library of 8 overlapped amplicons (10 kb each) for my 70 kb gene. I think i can use it and then with biorubter or covaris I can generate fragments with 200bp lenght for ePCR and sequencing afterward. What do you think....

herrroaa 07-25-2011 05:36 AM

Hi All,
How can I ligate ion torrent barcodes to my DNA fragments.
Regards,Tarek


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