Hi everyone,
We are trying to do a pretty simple restriction digestion but are having some issues and think it might be due to the plasmid we are trying to digest. Briefly, the plasmid is about 2200 bp big and was obtained through transformation of TOP10 cells and subsequent Miniprep with a Qiagen kit. We have tried single and double digestions with several different enzymes that are supposed to digestion this sequence and observed that none of the digestions seemed to work as well as that the undigested plasmid showed up as a band equivalent to a 2200bp long linear DNA
We are now wondering whether it is possible that we obtained a linearised or denatured plasmid by our method of plasmid purification. In the manual of the kit is reads "Long exposure to alkaline conditions may cause the plasmid to become irreversibly denatured. This denatured form of the plasmid runs faster on agarose gels and is resistant to restriction enzyme digestion."
Does anyone know how much faster such a denatured plasmid is supposed to run? Might it just run at the speed of a linearized plasmid? Or is it possible that we obtained a linearized plasmid through some other way that is not tied to denaturation because of alkaline conditions?
a) Include the PB wash step while extracting the plasmid. Nucleases in your prep could explain your results.
b) Sequence your prep to see if you are working with the correct plasmid. Try diagnostic or preparative PCRs and send the PCRs for Sanger sequencing to cover the entire 2.2kb plasmid. Alternatively, do a MiSeq on your plasmid and de novo map the sequence.
c) Try a different kit like Zymo's.
Hi Sigrun, It sounds to me like you are denaturing that plasmid and it is resistant to the restriction digestion. When carrying out the mini prep, how long are you leaving your cells in the P2 buffer before the addition of N3?
Ideally you want to add P2, invert the turn several times and then immediately add N3. It sounds like a preparative error. Also try using fresh mini prep buffers - they could have been contaminated. As Krishna has said, use PB buffer also if the strain is not like DH5a and has the endA+ gene (Check the info for TOP10).
Also just as a sanity check, the plasmid vector has an antibiotic resistance marker and that is selected for in the transformations, and the cells grow fine?
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