It may be possible that the amount you extracted was not enough to be
detected by ethidium bromide but would suffice for PCR. So far I have
not had any problem extracting DNA from a polyacrylamide gel.
Errol
On Fri, 25 Aug 2000 10:02:22 -0500, Lise Larsen
<larsenli at missouri.edu> wrote:
>I've tried the Qiaex II Gel Extraction Kit (Qiagen) with good results.
>Thanks for the response I've received.
>>To Vince Mulholland: My gels are stained with ethidium bromide. I tried to
>concentrate the DNA extracted by freeze/thawing and run it on an agarose gel
>without prior reamplification, just to see if I got any DNA out of my
>extraction. I didn't so I concluded that the problem was the extraction
>rather than the following PCR amplification.
>>Lise
>>Vince Mulholland wrote:
>>> Lise,
>>>> Rather than your extraction method, it could be to do with the staining
>> method you use. Do you silver stain your DGGE gels? If you silver stain,
>> do you use a stop solution which contains acetic acid? The acetic acid,
>> after silver staining, seems to render DNA refractory to amplification
>> (presumably due to nick of the strands).
>>>> Vince
>>>> Vincent Mulholland,
>> Senior Plant Pathologist,
>> Diagnostics & Molecular Biology Section,
>> Scottish Agricultural Science Agency,
>> East Craigs,
>> Edinburgh EH12 8NJ, U.K.
>>>> URL: http://www.sasa.gov.uk/>> E-Mail: Vince.Mulholland at sasa.gov.uk>> Tel:+44(131)2448845 Fax:+44(131)2448912
>>>> ___________________________________________________
>> How do you extract DNA from a polyacrylamide gel?
>>>> I run DGGE gels and need to excise the bands for PCR reamplification and
>> sequencing. I've tried cut out bands and freeze/thaw them ×3 in water
>> but didn't get any product after the following PCR. Please, let me know
>> if you know of a good method to recover the DNA.
>>>> Thank you
>> Lise Larsen
>>>> ---
>>>> ---
>
