To DNA sequencing experts!

Mr V. Schoenfeld DOM vs10005 at crc.ac.uk
Tue Dec 20 07:24:14 EST 1994


In article <3d5ag5$8vq at mark.ucdavis.edu>, szcooley at dale.ucdavis.edu (Michael Cooley) writes:
> Mic Chaudoir (mic at nwu.edu) wrote:
> : 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
> 
> 

Don't bother caesium prepping your DNA! 
Honest, the good old alkiline lysis mini works fine and it's dirt cheap.
Take 3ml LB O/N and resuspend in approx 40 ul TE. 
Take 18 ul DNA + 2 ul 2M NaOH, let denature and EtOH precipitate. Wash and resuspend in a total
10 ul which includes primer and annealing buffer. Follow the protocol for the rest.
You said your co-worker used the same alliquot as you did, so your DNA is probably OK anyway!
Has he left the template/primer to anneal from 70 C to RT slowly enough? Has he denatured the
final product JUST BEFORE loading on the gel? They're the usual cock up points in the protocol.
Good luck.



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