Plasmid DNA Sequencing with Universal Primers - (Mar/08/2007 )

Hi all,

I've been having some trouble with sequencing. The gene I want is about 1.8kbp, but I've got nothing reliable after 2 trials with the primers I designed. I was told to try T7 forward and M13 reverse, but I'm getting even uglier results.

Since I'm brand new to this, I am wondering what the common issues to sequencing are, and what are some of the things I should keep an eye out for? Any help would be appreciated.

Dubs

Ok, now when you say your results are ugly and unreliable, what exactly do you mean? What do your chromatograms look like? Any way of showing a picture?
What plasmid are you using? How much DNA is in each reaction? What are your cycling conditions and purification procedure?
Some common problems -
Low peaks, sequence gets scrambly quite quickly, peaks have smaller peaks underneath them - not enough DNA.
Peaks are an OK height, but not clear, and it looks like there's 2 chromatograms, one on top of the other - mixed template. You either have 2 plasmids or 2 primers.
Big red peaks obscure some of your sequence - residual EtOH or other impurity.
Peaks have a 'lag', ie last peak runs into next peak and sometimes obscures it - rare problem, using less DNA can help.
Sequence is fine for a time, then abruptly goes to crap - long run of bases can cause this to happen.
Big peaks for a short time, then nothing - too much DNA.
Nothing - bad primer, loss of DNA, bad cycling conditions.
If your plasmid has a M13 reverse binding site, it should work like a charm, so something isn't right.
By the way, trying to sequence 1.8kb in one hit is a big ask, even with the improved DyeT thats around these days. Are you perhaps expecting too much...?

With bad results from multiple trials of different primer sets, I'd be looking at template purity issues, though it's a tough call with out seeing the trace. How did you isolate your plasmid DNA?

the problem is probably due to the purity of your plasmid. The DNA has to be the cleanest you can possibly get it to be. No RNA , no proteins, removal of phenol must be complete, if your extraction protocol used PEG percipitation, make sure you have removed the PEG.

The easiest way to remove PEG is to ethanol percipitate the DNA, followed by resuspension with TE, followed by another round of ethanol percipitation. I find this is the only sure way of removing PEG, as washing with 70% EtOH does not gurentee reliable removal of PEG.

Now that said, I find the sequencing reads are better if, the template DNA is suspended in water (as opposed to TE)
Also use of sequencing buffer is a must for reliable results.

The next important thing the amount of DNA and Big Dye terminator in used. The amount of DNA I use is about 400- 500ng.. although the amount used varies with the size of the template. On the matter of Big Dye more is not better. Do not use too much Big Dye.. you get too much product, which won't suspend uniformly and clogs up the needle and capilaries of the sequencing machine... giving bad sequence reads.

I use 1ul for plasmid DNA eg read lenghts of 900bp+. 0.5ul for PCR prdocuts.. read lenghts around 500bp.
Final reaction volume 10ul.

And finally the clean up.
THis is very important too. If you clean up your own sequencing reactions, this is another point that could ruin your reaction.
Salts - this must be removed very throughly. Fail to do this right and your sequencing read will look bad.

I percipitate my 10ul reaction sample by first diluting it with 90ul of sterile distiled water and 1ul Pellet paint (basically coloured dextran that helps pulls down the DNA into a nice colour pellet). Add 10ul NaAcetate, then add 300ul ethanol.

Put the mixture in the freezer (-20ul) for at least 30mins.
Spin down. Discard supernatant
Wash with 200ul 70% ethanol. Discard supernatant.

Dry at 37 Celsius for 5min... important to avoid over drying, you will get no read if the DNA is dried to an insoluble pellet.

And its down. It takes some practice.