2 replies to this topic

[Jeronimas]

Hello,

   I perform a PCR amplification with BSP primers, which have a M13 tail added to a 5' ends. It is a hemi-nested PCR, first round normal BSP primers, second round both primers with M13 tails, and forward primer is a bit closer to the reverse than in the first round.
  The amplicon is about 300 bp, and shows a sharp band in a electrophoresis gel. However, when I try  direct sequencing for this product (with M13 seqencing primer), I always get unacceptable sequence (e.g. photo attached to the post). It is very confusing, as I succeeded to get good sequences from another 4 amplicons this way. Why is this one different?
I use "Genetic Analyzer 3500" from ABI and BigDye seqencing kit. Any insights are highly anticipated.

Jeronimas

Attached Files

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[methylnick]

looks like you have mispriming occuring or you have multiple amplicons in your sample for direct sequencing.

Ar eyou amplifying a family member of a large gene family?

Are you trying to sequence through a repeat, or long stretch of one particular base?

[Jeronimas]

No the seqence I amplify does not belong to a large gene family, it is a gene promoter region.

Yes, there are couple of long streches of T, two of them >20 bp, couple of others >10 bp. But that was also the case in other 4 amplicons.

Can mispriming really occur in sequencing reaction with M13 tails? And how would you suggest to solve multiple amplicon problem if that might be the case?

grateful for your answer,
Jeronimas

methylnick, on 07 October 2010 - 02:31 AM, said:

looks like you have mispriming occuring or you have multiple amplicons in your sample for direct sequencing.

Ar eyou amplifying a family member of a large gene family?

Are you trying to sequence through a repeat, or long stretch of one particular base?