Hello,
I performed a single digestion on my plasmids (~6.5kb) using BamHI. The total reaction volume was 10µl (containing 0.5µl(10U) BamHI) and the samples were incubated at 37° for 1hr.
After the digestion, I stored the digested samples at -20°C and run the DNA gel electrophoresis on the next day to check the samples. I used 1% agarose gel and 1X TAE buffer ,and I run the gel for 45min at 100V. (The dye was DL5000(6X) FluoroDye, smobio )
My expected result is: only 1 band should appear in lanes containing completely digested sample (~6.5kb but higher position because of its linear form)
However, the result of the gel electrophoresis (as the figure below) showed smear bands and some of the samples seems to be incompletely digested. In one of the digested samples, there are 2 bands on gel, one is obvious at 12kb and another is above 12kb , no 6kb band was shown.
I'm wondering why? Did my samples ligate with each other?
My questions are:
1) What may cause the smear band or the band shifting in gel?
I was thinking about the possibility of BamHI binding to DNA, DNA overloading, wrong RE digestion condition or electrophoresis condition...etc. Should I inactivate the enzyme by heating after digestion or before electrophoresis?
2) Why are my samples incompletely digested?
Did I set the reaction condition wrong?
Sorry for asking lots of question, and thank you so much for answering!
I think there was problem with your Plasmid isolation. Run plasmid only for the first time, then after restriction enzyme digestion run both undigested as well as digested plasmid. Make sure you load 1 microgram of plasmid in each well. Sometimes smearing is due to problem with pH of the buffer.
Rajeev Kumar Mishra,
Thank you so much for your answering! I think I need to isolate my plasmid again and run the gel as your suggestion. Thanks again!
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