To DNA sequencing experts!

Mic Chaudoir mic at nwu.edu
Fri Dec 16 16:24:35 EST 1994


In article <1994Dec10.030247.20311 at news.snu.ac.kr>,
jongslee at cd4680.snu.ac.kr (JongSub Lee) wrote:

>  Hi experts
> 
> I wanna ask you something on sequencing.
> 1) When my co-worker sequenced the same DNA sample, his x-ray film did not
> show any bands at all sometimes and did show good bands sometimes.
> He did sequencing with exactly same DNA sample aliquots.
> Why did the results?

My guess is technique.  The dsDNA sequencing techniques are not trivial,
and inconsistent results are not uncommon.


> 2) On the sequenced bands, there are some ladders on all 4 lanes. There
> were no so many ladders but some ladders on the film. We cannot read the
> sequence around the ladders. How can I avoid that? 
> Is the sample quality problem? or primer-annealing problem?

I am not sure what you mean, exactly ?  If you mean that you simply had a
latter of bands in the lane, well, this is commonly seen as well. My guess
is that it would be caused by bad technique and/or not-so-clean DNA

Here are tips that we use for good results:

1)  Always use Cesium Pure DNA.  This gives the most consistently good results.
2) Make sure that all of your reagents are fresh
3) Do not over-extend.  Watch your times carefully.
4)  Pipette all mixtures with great care, avoiding bubbles


Hope this helps.  A better description of the second problem would be
helpful, though.

-- 
mic
mic at nwu.edu
 
"It [PowerPC Mac] won't have any effect at all on Intel machines.
They will continue to plod along, running the same clunky Windows              and wretched DOS drivel they always have.  The PPC will affect Intel PCs the same way an SR-71 Blackbird affects a dairy cow."
                     Robert Rhode, in comp.sys.mac.advocacy



More information about the Methods mailing list