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Old 09-14-2011, 12:47 PM   #1
soleulloa
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Location: Chile

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Question Trial PCR failed

Hi all,

I'm doing a mate-paired library for SOLiD 5500.
When I run the E-Gel to confirm a 300 bp fragment, the result was 200 bp.

When I did the Nick translation reaction, the protocol suggests 10-11 min and I set 10,5 min. All incubations and stop reactions were very very strict, as the protocol says.

Can I use this library to continue the experiment?
Can someone help me?

Thanks,

Soledad.
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Old 09-15-2011, 04:42 AM   #2
soleulloa
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Pleeeeeeeeeeeeeeeeease
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Old 09-18-2011, 09:51 AM   #3
pmiguel
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Hi Soledad,
We have had samples before that required a longer extension time to reach the desired size. So we tried a few longer extension times. On one library I seem to remember we went 14 minutes.

We got so tired of not getting the right size range, we just did several time points for each and pooled them. Not ideal, because the resulting smear covers a wider size range, but better than having to repeat the entire library construction procedure. Here I gave a little more detail:

http://seqanswers.com/forums/showthread.php?t=8912

and lots of background not directly related to your issue:

http://seqanswers.com/forums/showthread.php?t=5626

--
Phillip
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Old 09-20-2011, 11:02 AM   #4
soleulloa
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Quote:
Originally Posted by pmiguel View Post
Hi Soledad,
We have had samples before that required a longer extension time to reach the desired size. So we tried a few longer extension times. On one library I seem to remember we went 14 minutes.

We got so tired of not getting the right size range, we just did several time points for each and pooled them. Not ideal, because the resulting smear covers a wider size range, but better than having to repeat the entire library construction procedure. Here I gave a little more detail:

http://seqanswers.com/forums/showthread.php?t=8912

and lots of background not directly related to your issue:

http://seqanswers.com/forums/showthread.php?t=5626

--
Phillip



Thank you Phillip!!! I'll try to do using more time for nick translation
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