ion torrent

[herrroaa]

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]

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]

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]

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]

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]

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