Problem with EcoR-1 .
I have been trying to insert a gene of interest in pcr4TOPO vector. Vector insert joining and the transformation was carried out as per the guidelines in the protocol (Invitrogen ). The white colonies which were resistant to ampicillin (transformants) was selected and plasmid was isolated by NoLid putrification kit . I am using EcoR-1 restriction activity to confirm insertion of PCR product to the TOPO vector (TA cloning) The incubation time for restriction activity was 4 hr and in agarose gel running the undigested plasmid (control) give different forms of plasmid and in digested sample the plasmid with insert is fully degregated. For information the undigested samples also were incubated at 37tem.
My question is why do the plasmid with insert is fully degragated by EcoR-1.?
I appreciate your ansers and guide
thanks for your comment. however, our first two lines are coming from the same plasmid isolation.
You may know that EcoR1 will cut in every 5'-GAATTC-3' sequence!! So, you may have to use high resolution gel!!
thanks a lot Bannikov Artyom actually the competent cells are DH5 alpha
and i didnt use buffer AW , may be i will try one time after washing by AW buffer which could protect from contamination
From your gel picture, I see that there seems to be nothing in the lanes that contain digested plasmid. How much plasmid was put into the digest and how much did you run on the gel? You may not have enough DNA in the wells to be visible. What are the expected DNA sizes after complete digestion? If the sizes are smaller, you need to load more total DNA in the well to see the bands. I can also tell from your DNA ladder that fragments closer to the bottom of the gel are less visible than those near the top. If you are putting DNA stain into the gel itself, you may want to add this stain to the running buffer as well.
Did you use same amount of DNA in undigested and digested lanes? It looks like that the digested lanes the DNA is all gone. Are you sure you used the tube contains EcoRI restrcition enzyme as it supposes to? Or some other enzymes such as DNAse I?? Or your EcoRI tube comtaminated with DNAse?
Use a positive control. Take plasmids somewhere else (may be from a commercial kit, not your miniprep) and digest them. Run a gel with digested sample and undigested sample to compare. Test both the EcoRI from your lab, and EcoRI from another lab.
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