Bad sequencing result - (Aug/17/2006 )

Hello everybody,

I´m trying to amplify a 1,350bp fragment for a bacterial genomic DNA (16S rDNA) using universal primer 27F and 1492R. I got the problem about really low recovery of DNA concentration after cleaning up. I use the purification kit from Promega Co., and measure DNA concentration by using nanodrop. Then I tried to get enough DNA concentration (37.5 ng/ul) by using 8 gel bands (they are from eight 50-ul PCR products) and spinning them through a cleanup mini column. Then, I sent all samples to sequence.

However, I got the problem again about bad sequencing result . There are several peaks took place in the same time, so it can not justify what it is. I don't know what is wrong with my samples. I did and got this kind of result twice.

Well, some sugestions please?

Thanks.

Hi Jeanie!

Did you do the PCR for sequencing? Or was it done at the place you have sent your samples to?

If you did it the sequencing PCR the way you described it, the first thing is that you hopefully used one primer per reaction... it's not quite clear if you did that.
Using both primers would cause a mixture of peaks in your result.

Or maybe (if you used one primer) the primer that has been used for sequencing is able to bind at several sites of your template.

But it also could be that you have more than one template in your reaction...? This can happen when your DNA isn't clean enough or if you got two plasmids in miniprep for example.

Well, or maybe the clean-up after the sequencing PCR didn't work. If salt and leftover primers aren't removed there can be additional signals.

How high are your signals by the way?
It also can be that there is only background and your reaction failed or you lost the sample in clean-up.

Hm... there are quite much possibilties... maybe you can show me/us your peak diagramm?

Greetings and best wishes!
Chakchel

P.S.: Maybe those websites can help you, too!

http://www.nucleics.com/DNA_sequencing_sup...leshooting.html
http://www.edgebio.com/tech/tsg/Sequencing_tsg.html

Hi
Please can you upoad a figure of your electropherogram ? Anyway it seems to me that the PCR is far to be perfect and then that´s why you do not get any good sequence. Try to clone the PCR product you obtain and work with plasmids. Second idea (if you wanna avoid this) change your primers are not working for PCR, probably they will not work for the sequence either.

Micky

you're not adding both primers to the sequencing reaction, are you? the very first time I sequenced I had made that mistake...too busy with PCR-mode and hadn't really thought about how sequencing happened...felt stupid but most importantly quit making the mistake

of course, it's possible you're getting two products

here's a linkthat may help you

Hi ,

Thanks all you gave me suggestions and links. I already read the sites and now I am back to the starting point to see whether my samples are purified or contain more than one species.

Jeanie

If there are only a few peaks with multiple bases, then you could be seeing variation in the 16S sequences at different places in the genome of a single organism. it is not uncommon to have site-specific variation in the many copies of the 16S gene. Cloning the PCR product and picking single colonies would give you clean sequence for a particular 16S site (you don't get to know which one).