sequencing problem - (Apr/08/2008 )

We run several genes PCR (9 genes).
We purify PCR products from agarose gel (1.5%) with Mo Biol DNA gel extraction kit.

We then sent the purified product with one of the PCR primer for sequencing (Genewiz, CA).

Except one gene, all the other genes did not produce any result from sequencing, and company explains it due to no priming.

I then take the left over stuff from gel purification do secondary PCR.
On gel I run both secondary PCR product and left over purified product.

I see clear single clear band for gel purify product, and all secondary PCR produce desire product.

During troubleshooting with Genewiz, they and I both measure OD for the concentration I sent. Although we come out a large differences in DNA concentration (150 vs. 33 ng/ul), we both agree there is sufficient DNA template (purified PCR product) for sequencing reaction.

I understand that sequencing and regular PCR are 2 similar but very different things.
There is 1 gene come out great result; thus overall, the systems from PCR to sequencing should work.
But come on, 1 out of 9 works, there is something wrong.

What I miss here?

What problem can be that cause no priming?
What else I can do to have sequencing result?

We run several genes PCR (9 genes).
We purify PCR products from agarose gel (1.5%) with Mo Biol DNA gel extraction kit.

We then sent the purified product with one of the PCR primer for sequencing (Genewiz, CA).

Except one gene, all the other genes did not produce any result from sequencing, and company explains it due to no priming.

I then take the left over stuff from gel purification do secondary PCR.
On gel I run both secondary PCR product and left over purified product.

I see clear single clear band for gel purify product, and all secondary PCR produce desire product.

During troubleshooting with Genewiz, they and I both measure OD for the concentration I sent. Although we come out a large differences in DNA concentration (150 vs. 33 ng/ul), we both agree there is sufficient DNA template (purified PCR product) for sequencing reaction.

I understand that sequencing and regular PCR are 2 similar but very different things.
There is 1 gene come out great result; thus overall, the systems from PCR to sequencing should work.
But come on, 1 out of 9 works, there is something wrong.

What I miss here?

What problem can be that cause no priming?
What else I can do to have sequencing result?

My direct sequencing never worked, I always cloned it etc. But it was another problem.
How specific is the PCR? Are you sure to have amplified the correct fragment? Perhaps a restriction cut of the PCR product helps, if you have information about the sequence.
Are the primers suitable for sequencing?

Hey,

There are many things to consider when direct sequencing your PCR product. I know because this is my first year in molecular as a new grad and I am doing a lot of this stuff independently. It’s been a struggle but I’ve learned a lot. J

First off, how specific are your primers to your target. I’ve been doing a lot of degenerate PCR, which opens up another range of considerations. But if your primers are not very specific to your target then they could have trouble with the sequencing reaction.

What is the annealing temperature of your primers in comparison to that of the sequencing facility? My facility anneals at around 50C and I’ve had primers that anneal at 47C for PCR. If you trying to sequence, it may be too warm for your primers to bind.

Don’t send too much DNA for sequencing. Example, I have a ~800bp fragment and my facility tells me to send NO MORE than 100ng or it can clog the column. Small fragments for direct PCR sequencing don’t require a lot of DNA.

Make sure you PCR clean-up is really good. I use the Qiagen PCR clean-up kits. Salts and other stuff can mess up the sequencing reactions. My sequencing facility requests that samples be sent in molecular H20.

Make sure your primers are not expired. I was having trouble with sequencing because I inherited old primers. Things were working fine and one day just flopped. After I got new ones, things went back to normal.

I’ve done a lot of independent learning about this stuff online.

Here is a link about direct sequencing PCR products this will repeat/reiterate a lot of what I just mentioned

http://seqcore.brcf.med.umich.edu/doc/dnaseq/pcr.html

Here is a link that explains DNA sequencing.

http://www.roswellpark.org/Site/Research/S...quencing_Basics

I hope this helps. Its better to ask questions now than reinvent the wheel. Stick with it!