Question: Sanger Sequencing showing too much background noise?
0
gravatar for sandKings
7 months ago by
sandKings10
sandKings10 wrote:

One of my colleagues is trying to do Sanger Sequencing. In her words:

"I am isolating DNA from tissues, amplifying the gene of interest, purifying it and sending it for sequencing. However, I get high-intensity peaks with lots of background: see peaks https://imgur.com/a/R1mwp

Do you have any suggestions I can try? I measure DNA conc. using Nanodrop before sending it to them, but it turns out higher than what I measured and gives high-intensity peaks. Although I see only one band corresponding to the desired product size. Can this be a primer issue? "

Can someone please suggest her steps to troubleshoot this.

Thanks!

sanger sequencing • 297 views
ADD COMMENTlink modified 7 months ago by Michael Kosicki80 • written 7 months ago by sandKings10

This is not strictly a bioinformatics question. You may want to post this over at SeqAnswers.com.

ADD REPLYlink modified 7 months ago • written 7 months ago by genomax56k

Right! Sorry about that.

ADD REPLYlink written 7 months ago by sandKings10
1
gravatar for c.chakraborty
7 months ago by
c.chakraborty90 wrote:

Is the DNA uniformly dissolved? Usually, if the DNA is not uniformly dissolved it might be too concentrated at the bottom of the tube. You can try to add more water, pipette it up and down vigorously quite a number of times, and dissolve the DNA at 37 degrees for 30mins to 1 hour and then recheck the concentration. Another thing I can suggest is that you do the PCR and purify the DNA again, before sending it for sequencing. If the previous batch is not clean enough (still contains some residual salt or oligos), then sequencing will give you noise. Also, are you sending them your own oligos for sequencing or are you using custom oligos from the company? If you are proving oligos for sequencing, I would suggest make a fresh new batch this time, maybe the oligo solution is dirty.

ADD COMMENTlink written 7 months ago by c.chakraborty90

Thanks C. She is using her own oligos and she said she'd make a fresh batch as well as try purifying the DNA.

ADD REPLYlink written 6 months ago by sandKings10
1
gravatar for Michael Kosicki
7 months ago by
Wellcome Trust Sanger Institute
Michael Kosicki80 wrote:

If I understood correctly, they have performed a PCR on the material and then Sanger sequenced the product.

1) The sequencing service provider usually specifies, whether the product needs to be purified (e.g. using beads or columns) or whether they can do it for you (usually for a fee) and how much to send.

2) Sometimes the primer is a problem, try another one. It may be primer itself or the sequence that is downstreams of the primer - e.g. a run of Ts can cause problems.

3) Concentration is rarely an issue, unless they've used Nanodrop on the product PRE-PURIFICATION, in which case they're measuring the primers and the pure nucleotides. Use Qubit, Bioanalyzer, Picogreen etc, if you need to work with the unpurified product or purify and do the Nanodrop.

ADD COMMENTlink written 7 months ago by Michael Kosicki80

Hi Michael, thanks so much for the feedback. I know she's using nanodrop but am not sure if it's before or after purification. I'll confirm

ADD REPLYlink written 6 months ago by sandKings10
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