Question: sanger sequencing problems
0
gravatar for Elnaaz
4.8 years ago by
Elnaaz40
Austria
Elnaaz40 wrote:

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 .

sequencing snp • 2.1k views
ADD COMMENTlink modified 4.8 years ago by Kizuna780 • written 4.8 years ago by Elnaaz40

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

ADD REPLYlink written 4.8 years ago by Elnaaz40

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"?

ADD REPLYlink written 4.8 years ago by Michael Dondrup46k

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

ADD REPLYlink written 4.8 years ago by Dan D6.8k
0
gravatar for Michael Dondrup
4.8 years ago by
Bergen, Norway
Michael Dondrup46k wrote:

Primer dimer ?

who sequences primers anyway? possibly not related to bioinformatics.

ADD COMMENTlink modified 4.8 years ago • written 4.8 years ago by Michael Dondrup46k
0
gravatar for Kizuna
4.8 years ago by
Kizuna780
France, Paris
Kizuna780 wrote:

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

ADD COMMENTlink written 4.8 years ago by Kizuna780
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