Problem with restriction digestion of agarose-embedded DNA (View forum version)


Posted 22 December 2011 - 06:44 AM

Hi all.

I am on BAC construction way and having a problem with partial digestion of agarose-embedded DNA with BamHI. The agarose plugs were digested with series of BamHI from 0 to 10 U in 37C for 1 hour and PFGE. The problem arose when I could not get smear band as expected even with the plug that treated with 10U BamHI. I am sure about RE, buffers and other PFGE condition and the only thing I think should be problem is the plug. After pre-electrophoresed to remove the small fragments, I stored plug in TE (20:50) before make partial digestion. I read that high concentration of EDTA should inhibit the RE's activity. Is it the reason why I got problem???

Anyone has the same experience please help me
Thanks in advance


Posted 22 December 2011 - 06:50 AM

Plugs must be washed free of EDTA prior to digestion. This is usually done by washing 2x in large volumes of restriction enzyme buffer for several hours or overnight. A wash at elevated temperature (37-50) will be faster, but make sure you don't melt the agarose. The screened caps for 50 ml tubes sold by Bio-Rad are very useful for these washes and digestions.


Posted 22 December 2011 - 06:57 AM

Thank phage343 for immediate response.
Yes I performed 2x1hour washing of plugs in RE buffer before incubate in RE buffer + RE 4h to diffuse RE into plug. But the result was as I stated. I wonder that a washing step with TE (10:1) or TE (10:0.5) before wash with RE buffer is need or not.

Do you have any advice?
Thanks a lot


Posted 22 December 2011 - 08:25 AM

Are you certain your genomic DNA can be cut with BamHI? I had a problem once with a strain that had methylated GATC sites, inhibiting BamHI cutting completely. I'd check with a gDNA sample cut on an ordinary low percentage agarose gel, just to make sure.


Posted 24 December 2011 - 07:40 AM

I don't thing BamHI is a rare restriction or difficult to cut site in plants and this is one of REs used in BAC construction KIT as far as I know. Therefore I think the problem is on my plugs or more precisely my HMW DNA preparation have trouble. I will try with gDNA first as your suggestion. Hope that it will work.
Thank you and Merry Christmas