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Old 06-27-2011, 05:12 AM   #1
herrroaa
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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
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Old 07-02-2011, 07:44 PM   #2
krobison
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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?
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Old 07-04-2011, 12:09 AM   #3
herrroaa
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Quote:
Originally Posted by krobison View Post
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.
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Old 07-05-2011, 12:20 PM   #4
krobison
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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.
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Old 07-06-2011, 06:01 AM   #5
herrroaa
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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....
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Old 07-25-2011, 05:36 AM   #6
herrroaa
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Hi All,
How can I ligate ion torrent barcodes to my DNA fragments.
Regards,Tarek
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