Restriction problems - (Oct/08/2012 )
I have been trying to clone my insert into binary vector. However after trying for 15 days I find that the problem is restriction of plasmid. I am unable to restrict the plasmid and have also tried different restriction sites. I have used this plasmid earlier and it worked fine. I have tried restricting the entire cassette from the plasmid and I find that there is partial restriction.
The plasmid is ~16kb binary vector and I have cloned it in Dh5 alpha cells.
Has anyone face this kind of problem. If so please help me out.
Give us the details of your restriction digest. This is often caused when people do digests in low volumes where the majority of the liquid is uncut DNA. Dilution is your friend, and helps in reducing the effect of inhibitors in your DNA prep.
The restriction sites I have used are XbaI/Xho and StuI/XhoI. For cassette I have used HindIII HF and EcoRI and this shows partial restriction. I did use 1ug Plasmid in 50uL for all reactions. Also I gave it to my labmate to restrict in case I was doing something wrong. But he too could not get the restriction.
I do multiple mini prep to get the plasmid (16kb binary vector). I wonder if this is contributing to inhibitors. Should I do a midi prep.
You didn't say what the VOLUME of your DNA in your reaction was. A very common problem is doing reactions where almost all of the volume is prepared DNA, which very often has alcohol or Gu-HCl contamination. Try cutting 100 ng rather than 1 ug. Try doing the reaction in 200 ul rather than 50 ul.
The plasmid prep is by qiagen column and the DNA is in about 7-10uL. Rest is Restriction enzymes about 1-2 uL, Buffer, BSA and water. I will try a lower volume and let u know. Thanks for talking time to answer my query.