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Old 10-08-2014, 03:53 AM   #1
Psuedomonas
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Location: New Zealand

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Exclamation Formamide problems?

I've accidentally added formamide to my mixture before PCR (tube contained template, primer, polymerase...) I had mistakenly grabbed a tube of formamide instead of my BigDye buffer and added that to my mix, which I was then going to put through PCR and then Sanger Sequence on an ABI 3730.

I'm interested to know what would happen if I was to PCR this formamide-containing mix, and if I would get a sequence, a formamide profile, or a failed reaction?

(My guess is that a formamide profile would appear after Sanger as it would inhibit annealing to ssDNA during PCR?)

Some of the literature I've seen appears to say that formamide helps sequence GC rich templates, but in general, has anyone had this happen to them? Does anyone know what a formamide "contaminated" sequence looks like?

I'm really worried as I really don't have a lot of this template and it took quite an effort to get my plasmids.
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Old 10-08-2014, 04:24 AM   #2
nucacidhunter
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You can add water to your miss-assembled reaction to total of 300 ul and clean it with a PCR clean up column and set up a correcr sequencing reaction.
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Old 10-08-2014, 04:38 AM   #3
Psuedomonas
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Ah, but hypothetically what would happen if one were to sequence that? Would that actually produce sequence?
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