I am working with Staph. epidermidis isolates two years ago I carried out a cloning experiment on my Staph. isolate amplicons and made a glycerol stock of the recombinants stored away in -80 freezer. But after two tears I carried out a restriction digest on them and I cant see the two bands on the gel electrophoresis signifying the presence of my recombinants. can anyone please advise me on this. many thanks
Did you plate your strains out on selective media for the plasmids to be sure that only the recombinants grew? I've stored recombinant strains for decades at -80 without losing the plasmids.
Thank you for the answer, yes I did grow them on antibiotic selection plate but after digestion with restriction I fail to identify my insert band can only see the plasmid band. Thank you
Maybe the stock you froze away was mixed with recombinant and parent plasmid. Streak it out and try mini prepping a bunch of colonies, or try colony PCR
I have done colony PCR a couple of times with the glycerol stock using insert primers and can find the fragment size for my insert. But whenever i try digesting with the restricion enzyme i cannot find the insert band accept that of the plasmid. Strange though is the fact that two years ago when i carried out the cloning experiment i did plasmid extraction from those same stock and did restriction digest on them and found the double band of the olasmid and insert.
I find it very doubtful that a -80 freezer stock has changed with regard to which plasmid is being carried.
It is a good sign that colony PCR with the stock does show that some plasmids still carry the insert. I reiterate my suggestion that you just have a mixed stock (perhaps majority without insert) and if you look at a bunch of individual colonies from the freezer stock you are likely to find a pure stock of the plasmid you want.
To determine the cause of the absence of the recombinant plasmid, you may consider the following steps:
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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