Hi,
I was given glycerol stocks of DH5-alpha containing a plasmid of interest. I plan on using this plasmid in a site-directed mutagenesis experiment using Stratagene, who suggests the plasmid be mostly in supercoiled form. However, a plasmid prep revealed many large bands on a gel (see attached image), so large in fact that they must correspond to multimers (i.e. they do not correspond to linear or open circle conformations). I do not think the bands are from a contaminant but I am not 100%. I do no think this plasmid template will work well in a primer-based PCR mutagenesis experiment. What should/can I do in order to get mostly supercoiled plasmid? Additional preps using the same glycerol stocks will produce the same multimers, right? Should I purchase a new glycerol stock from a company? (The stocks I was given were generated in-house). Could I gel-purify the supercoiled and retransform? Any help is greatly appreciated!
if you have not used a commercial kit for your plasmid prep then I must compliment you on one of the cleanest plasmid preps that I have ever seen. Now the different bands that you are observing is how you should see your plasmid after alkaline lysis. the big prominent bands that you see running above your expected size are indeed the supercoiled form and then faint bands at the correct size are the liner form. is there a particular reason that you expect these to be multimers? A simple restriction digest would answer this question.. In fact you should look at the original sequencing data and also do a restriction digestion to confirm your plasmid as you were given the glycerol stock by someone else, that's just GLP. Again after doing a confirmation test (PCR, Sequencing or restriction digestion), I would confidently use the plasmid, for mutagenesis, that you have prepped
If you really do have concatenated plasmid for some reason, then you should be able to decatenate it by treating it with topoisomerase IV. Here are 2 commercial sources:
https://www.topogen.com/topoisomerase-enzymes/e-coli-topoisomerase-iv.html
http://www.inspiralis.com/go/s_pneumoniae_topoisomerase_iv
Hi Jason,
I agree with previous comments. you should cut your plasmid with RE to confirm the insert .
I just want to add some points. First of all, the expected bands from your image is not supercoiled DNA, I belive they are just fragments. You have to run your gel with 10 Kb marker and without EtBr or any other intercalator. In bacteria, more than 90% of the DNAs are supercoil and 10% is nicked or linear conformation. When you use a intercalator they become more supercoiled. You could not see the differences between topoisomers. However, your problem is coming from your gel run. Please run your gel 45 volts for 3 hours in 1% agarose gel without EtBr. After that, you have to stain your gel by 1 ug/ml EtBr for 10 min. Let your execive EtBr wash away from the gel in dH2O for 30 mins. Check your band on UV transiluminator.
I hope this works
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