ion torrent

herrroaa · 06-27-2011, 06:12 AM

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, 08:44 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, 01:09 AM

Quote:

Originally Posted by krobison

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, 01:20 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, 07:01 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, 06:36 AM

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

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06-27-2011, 06:12 AM	#1
herrroaa Junior Member Location: stuttgart,germany Join Date: Jun 2011 Posts: 4	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

07-02-2011, 08:44 PM	#2
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	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?

07-05-2011, 01:20 PM	#4
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	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.

07-06-2011, 07:01 AM	#5
herrroaa Junior Member Location: stuttgart,germany Join Date: Jun 2011 Posts: 4	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....

07-25-2011, 06:36 AM	#6
herrroaa Junior Member Location: stuttgart,germany Join Date: Jun 2011 Posts: 4	Hi All, How can I ligate ion torrent barcodes to my DNA fragments. Regards,Tarek