Thanks in advance for any help

Posted 07 May 2009 - 03:43 AM
Posted 07 May 2009 - 05:24 AM
Hi could you tell us the nature of your DNA, is it genomic DNA or plasmid DNA.Hi I was just wondering if anyone could help me. Whenever I do a restriction digest all i get is a big smear. I have tried multiple different digests and they all result in a smear. To check for DNase I contamination i incubated my pDNA with water and the pDNA was not degraded. I then also incubated my pDNA with water and buffer and this results in a smear which suggests the contamination is in the buffer. I have tried the digests with many different buffers and all result in a smear. I have tried heating the buffer and pDNA at 75oC for 10mins to inactivate any DNase but it still results in a smear. I was wondering if anyone had any suggestions. Also is it possible that there is something else degrading my DNA other than DNase?
Thanks in advance for any help
Posted 07 May 2009 - 06:42 AM
Hi I was just wondering if anyone could help me. Whenever I do a restriction digest all i get is a big smear. I have tried multiple different digests and they all result in a smear. To check for DNase I contamination i incubated my pDNA with water and the pDNA was not degraded. I then also incubated my pDNA with water and buffer and this results in a smear which suggests the contamination is in the buffer. I have tried the digests with many different buffers and all result in a smear. I have tried heating the buffer and pDNA at 75oC for 10mins to inactivate any DNase but it still results in a smear. I was wondering if anyone had any suggestions. Also is it possible that there is something else degrading my DNA other than DNase?
Thanks in advance for any help
Posted 08 May 2009 - 12:48 AM
Posted 08 May 2009 - 06:05 AM
Thanks for your help. I am using pDNA and I am transforming it into DH5a cells. I suspect that the pDNA may be getting contaminated during the pDNA prep so I was thinking the best idea might be to do the suggested phenol/chloroform extraction. I was wondering if anyone had good protocol for doing this on my current pDNA?
Thanks again
Posted 10 May 2009 - 11:19 PM
Posted 19 June 2009 - 12:18 AM
Posted 05 July 2009 - 11:05 PM
I had the same problem...for me the solution was very simple...cleaning the gel chamber and fresh running buffer solved the problem. It could however also be a problem of the loading buffer...don't forget to consider this options, when having a problem like this...also using filtered tips could help...
Posted 30 July 2009 - 12:57 PM
Posted 04 August 2009 - 01:29 AM
Posted 04 August 2009 - 10:30 PM
Posted 30 September 2009 - 02:07 AM
Posted 24 September 2010 - 02:25 PM
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