Sequencing problem. Is it the GeneClean?

rmeck at icbr.ifas.ufl.edu rmeck at icbr.ifas.ufl.edu
Fri Feb 11 00:15:51 EST 1994


In article <94039.133242NORVALC at QUCDN.QueensU.CA>, <NORVALC at QUCDN.QueensU.CA> writes:
>   I've been sequencing PCR products for several years now and am continually
> frustrated by the lack of consistancy in my results.  At times I'll get perfect
> results and then for a period nothing seems to work.  I have tried manipulating
> many of the variables in the proceedure (I follow a standard protocol) and
> none of my variations seem to significantly affect my results.   Following the
> most recent derailment of my efforts I have tried using new reagents, new
> Sequenase, new isotope, variations of incubation times and temperatures, etc.
> I try all these variations in a controlled fashion.
>   Now I wonder if my problem is in the DNA cleanup stage.  Normally I harvest
> 
> a PCR band from a Nuseive agarose gel and use GeneClean II to clean up the DNA.
> I dissolve the agarose with the NaI, add glassmilk, use NEW wash to clean the
> DNA three times, and resuspend the DNA in a small volume of TA.
> 
>   My question is straightforward:  Have anybody else noticed that there are
> critical steps in the GeneClean protocol?  If so, what should I be focusing on?
> 
>   Thanks in advance for your help.  I will summarize and post any replies.
> 
> Barry Campbell
> Molecular Ecology Laboratory
> Department of Biology
> Queen's University in Kingston
> CANADA  K7L 3N6

Up to a few weeks ago I've used GeneClean too. But then I had severe
difficulties with cutting and especially ligation and transformation. 
When I scanned my DNA prep from OD200 to OD300 I noticed a very odd
profile and concluded salt contamination from New Wash solution. Anyway 
to cut a long agonizing story short I use now a different protocol
not GeneClean and I am happy with this, it is faster and more convenient then
glassmilk and involves cutting a trough in front of band fill it with PEG 15
percent and let the DNA migrate into it. Then Chloroform/Phenol extraction
EtOH precip and done. Method is published: Zhen and Swank. 1993. 
BioTechniques. vol 14 no. 6 page 894-898. 
Losses are almost 0.0 
Can send you copy of paper if reqested.

Reinhard
 




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