sanger sequencing problems

Question

[Elnaaz]

Dear all,

I have SNP in my samples after Illumina sequencing and then Designed specific primer to confirm it by sanger ,

I sent forward and reverse primer both to sequencing to make sure ( But Separately),

But after sequencing the chromatograms and sequecher show me different size for Forward (bigger than expected) and for Reverse the size is correct,I do not know why ? it is the base pair double than my expectation .

[Elnaaz]

The sequencing firstly was done by miseq . And for Sanger seq we did not have any dimmers or smears

[Michael]

Why don't you hear with the sequencing lab and the primer provider, I guess your seq lab is to blame? In the worst case just order the primer somewhere else if you don't trust the first providers, primers are cheap. Btw, where is "Austrila"?

[Dan D]

Re "Austrila": I was thinking the same thing--I just didn't want to be the guy who pointed it out. :)

Answer 1

[Michael]

Primer dimer?

Who sequences primers anyway? Possibly not related to bioinformatics.

Answer 2

[Kizuna]

In Sanger sequencing, you should have same results using the F and R primers.. F and R primers are used as a double check.

Are you sure that you are amplifying the correct genomic region? How big is you PCR product? It is prefered with Sanger to not have PCR products bigger that 300 bp.

Hope this helps,

kiz