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I am using CEQ 8000 genetic analyzer of BEckman coulter. some time i got good PCR amplification band on agarose gel. but sequencing results of that product with same primers appear very bad and most of the time even fail to sequence. what is the possible reason and what can be the possible solution. ?
please reply.Asif Ullah Khan
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This can happen if you have not removed all the PCR primers from the reaction. Also you can have mis-annealing products in the reactions. You can’t see these on the gel, but in total they will ruin your sequencing reaction.
The solution is to use internal primers for the sequencing reaction. it is never a good idea to use the same primers you use for amplification for sequencing PCR products.
Daniel Tillett
Nucleics Support
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