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Old 11-11-2011, 11:53 PM   #1
Isabel_Bodmer
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Location: Switzerland

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Default sequencing primer annealing

Hi everybody
I am new here and would like to first say thank you for this great forum. I'm looking forward to being active in it.
I have a problem with emPCR lib L. Everything worked fine until the very last step, the sequencing primer annealing. All our heating blocks were occupied so I used one of another group. I had problems with checking the temperature and it is possible that the temperature hadn't reached 65C yet when I put in my sample. At the end I figured it out and the temperature was right, but it's possible that it was only on 55C or so at the beginning.
Now I'm afraid that the primers may have bound nonspecifically or that it was only for some 2-3 minutes at 65C and not enough primers could bind to the DNA.
I would have enough sequencing primers for another annealing round. Do you think I could add some melting solution to remove all sequencing primers and the perform washes with annealing buffer and then again perform the sequencing primer annealing step?
I really hope you can give me some advice soon. Thanks in advance.
Cheers,
Isabel
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Old 11-18-2011, 11:30 AM   #2
Nicole 454 Sequencing
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Default

Hi Isabel,

I wouldn't proceed with sequencing until repeating the sequencing primer anneal step here. You can perform the Melt step as specified in section 3.6.1 of the emPCR Methods Manual and then skip ahead to the sequencing primer annealing (section 3.7).

Please don't hesitate to contact your local Roche representative with any further issues.

Nicole

Technical Support Scientist
454 Life Sciences, A Roche Company
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Old 11-18-2011, 12:20 PM   #3
Isabel_Bodmer
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Hi Nicole
Thanks for the advice.
I have already repeated the melting and annealing step and the sequencing worked fine.
Cheers,
Isabel
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