Problem with restriction digestion of agarose-embedded DNA - (Dec/22/2011 )
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
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.
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
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.
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