SSCP single strands

Bernhard Mayr ndxdbmay at rrzn-serv.de
Tue May 20 14:26:47 EST 1997


Your Name Here wrote:
> 
>   I am using SSCP to analysis for mutations in exons 5-8 of the p53 gene.
> I am denaturing my PCR product (1:2) with 95% formamide, 10mM NaOH, 0.5%
> of the dyes BB and XC, for 5minutes at 95 degrees C, followed by plunging
> the tubes in a 4 degree C water bath.
> 
>   I am seeing plenty of double stranded PCR product on a 6% polyacrylamide
> gel, however the amount of single stranded product is variable and
> sometimes there is none.
> 
> I would be grateful for any emailed advice regarding how to achieve
> efficient denaturation (without methyl-mercury or biotin) of my PCR
> product for SSCP analysis.
> 
>    Thanks in advance,
> 
>    Greg Jacobson
> 
>     gmj at waikato.ac.nz
> 
>    Greg

With SSCP a lot of steps a critical:

The PCR-product shouldn't exceed approx. 300-400 bp or you get multiple
conformations (=bands) that make gel-interpretation more difficult.
Sometimes it's useful to cleave your sample with a restriction enzyme in
2 pieces of different size prior to SSCP, which should give additional
bands in a different range on the gel.

Denaturing and cooling down should be highly reproducible, I use my
thermal cycler to 10min @ 95°C and cooling @ max speed to 4°C.

I dilute my samples 1:1 in standard sequencing stop buffer (95%
formamide, a litle bit of EDTA and a little bit of dye).

I get best results when running gels at a defined temperature, I use a
Hoefer device designed for protein gels with the gel fully immersed in
buffer that is cooled with a cryostat. Depending on the template I use
temperatures between 4°C and 25°C and for some of my templates  even +-
4°C make a lot of difference, you have to find out for yourself and for
your templates what's best.

I do not routinely use FMC's MDE gel although it gives great results (no
affiliation), but when using normal AA I use a ratio of AA to Bis-AA of
49:1 .

Hope this helps.
-- 
Bernhard Mayr
Medizinische Hochschule Hannover
Dept Clin Endocrinology
Hannover, Germany



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