Problem with restriction digestion of agarose-embedded DNA
Posted 22 December 2011 - 06:44 AM
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
Posted 22 December 2011 - 06:57 AM
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
Posted 24 December 2011 - 07:40 AM
Thank you and Merry Christmas