Sequencing reaction failure- Only Background - Sequencing reaction (Apr/27/2007 )

Dear bioforum members,

I’ve been trying to sequence several PCR fragments from a 10kb mutated plasmid and all I keep on getting is background on my chromatograph.

Here is what I did:

-Started by performing a rapid preparation of my plasmid extracts (from one bacterial colony each) for PCR purpose;

-PCR was performed for 9 samples;

-Purified PCR product with a DNA purification kit;

-Analyzed purified PCR product on an agarose gel, where distinct bands were observed;

-Sent the PCR product for sequencing with one primer per reaction(diluted the stock solution (1ug/ul) 1/15 times.)

Technician returns with a chromatograph with only background for all 9 samples.

I thought perhaps I mixed up the primers; so I sent it for sequencing again and still the same thing!

I know it’s not the machine because other people’s samples are sequencing well with the same run.

I’m at my wits ends on what the problem could be.

Does anyone have any suggestions? Help!


P.S- I used the same forward primer for a sequencing reaction a couple of months ago and it worked. I used a similar template a couple months back. The only difference is that the current template has a mutation site through a site-directed mutagenesis reaction.

Did you gel purify the bands, or just purify the PCR product directly? Short PCR products can be very abundant, but dim on a gel because they contain little DNA. You may have many more molecules of very short product than the desired product in your tube. Another way to fail is to use either too little or too much DNA. Too little fails to produce significant product -- the sequencing reaction is linear, not exponential in the number of cycles. Too much DNA yields only very short fragments, since all the dNTPs are used up early in the cycle. Pay attention to the amount of DNA -- it should be around 100 ng for a typical PCR product, less for short ones, more for longer ones (the molarity is what really counts).

I use qiagen miniprep kit for isolation of plasmid and sequence it right away (after checking concentration). There's no need to do PCR and all that. Probably other kits will work as well.

Check the concentration of your DNA before sequencing.

If you gel purify: that might well be the cause. UV-damage and remaining agarose can inhibit sequencing reactions... (A colleague of mine did it and asked "my group" (my supervisor and a lab technician are occupied with sequencing a lot) what went wrong, we just told her not to gel purify, she didn't and got good results).

Thanks a lot for your suggestions- Phage and Vairus

- I purify the PCR product directly and when I visualize it on an Agarose gel, I see heavy bands.

-The technician who performs the sequencing reactions says he is able to estimate how much DNA he uses by merely visualizing the bands on the agrose gel (I'll definitely have to check the DNA concentration before sending it for sequencing next time)

-I am currently not using a kit to isolate my plasmid- the reason being that I want to check if I have induced a mutation on the plasmid before purifying it with a kit. I, therefore, use a "rapid preparation of plasmid extracts" protocol, where I resuspend the bacterial colony in a mixture of TRIS 1 M, EDTA 0.25M, 3% Triton X-100 and autoclaved water. I heat this mixture for 10 min at 95 C, pellet the debris and use 4ul of the supernatant for the preparative PCR. I'm thinking perhaps the problem lies with this protocol?

Any thoughts? Thanks once again.