DNA SEQUENCING PROTOCOL - (Dec/25/2006 )
I have a simple question. For DNA sequencin I am using classical method for preparation of the sequencing mix.
After my PCR reaction, I visualize the bands on the agarose, and after that i am supose to cut only the wanted bands, not those resulting from primers or primer dimers. So, by now I am Ok. To extract the fragments from agarose I use ethanol precipitation with sodium acetate pH 5,2. My question is how many ml of this mix should I use, starting from lets sey 0,33mg PCR fragment in agarose gel. Becouse somtimes I get good yeld, but sometimes nothing. I dont think that i am makink some mistake in the rest of the protocol
I use big dye terminator 3.1. i have never run the product out on a gel, i just send it for sequencing. only 1 or 2 don't turn out (keeping in mind, i usually send 30+).
when looking at plasmid DNA, my reaction usually begins with 400-600 ng DNA for the PCR reaction. I ethanol precipitate everything in the tube.
I don't check my bands on a gel either but some people do to see how much crud there is and how intense the band is. Generally I use plasmid DNA for which I use 50 ng per sample for the dye termination reaction.
It depends on the length of your PCR product how much you have to add to your reaction: e.g.
0.5 kb, add between 8.1 and 33 ng of purified PCR product. The longer your sequence and if it's double stranded, the more you will have to put in.
To precipitate the DNA you have to add a stop/glycogen solution (2 ul 3M sod acetate, 2 ul 100 mM Na2-EDTA, 1 ul 20mg/ul glycogen) to your sequencing reaction (20 ul in case of Beckman starter kit) and add 60 ul 95% etho (-20C). After 15 min spin at 4C rinse twice with 70% etho. Let it dry out in a heating block at 56C and add your formamide (40 ul). Don't mix, just add the stuff and leave at +4 for 10 min. Then vortex and sequence.
hope this helps.
Aproximatelly 20ng of PCR product will work fine.