Biostar Beta. Not for public use.
sanger sequencing problems
0
Entering edit mode
5.7 years ago
Elnaaz • 40
Austria

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 .

SNP sequencing • 2.1k views
ADD COMMENTlink
0
Entering edit mode

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

ADD REPLYlink
0
Entering edit mode

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
0
Entering edit mode

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

ADD REPLYlink
0
Entering edit mode
20 months ago
Bergen, Norway

Primer dimer ?

who sequences primers anyway? possibly not related to bioinformatics.

ADD COMMENTlink
0
Entering edit mode
3.1 years ago
Kizuna • 780
France, Paris

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

Login before adding your answer.

Similar Posts
Loading Similar Posts
Powered by the version 2.3.1