We are trying to transformation about 3-4 months. I used different methods, different subsp. of E. coli (e.g.: JM101, DH5a, TOPO..) but with them I didn't find any success. I used electroporation technices but this time all the petri plates were covered with bacteria which is I confused more. After heat-shock I poured 50uL-100uL-150uL bacteria to the plates but some of them turned into nothing. Even if we did the successfull transformation, we picked colonies for the PCR tests, and we get better "negative" results.
Is there any suggestions? Thanks a lot.
hi,
which antibiotic did you take? big plasmids do not be anissue in principle, but if you have a high evasion pressure (long plasmids might trigger thi smore) cells try to get rid. if you use e.g. Amp you mit replace it with something more stable like Carbencillin
If after electroporation you are getting too many colonies, you could do further dilutions to get better isolated colonies. But in case these are not real, you should always do a control alongside with no added DNA to verify if everything is working or whether there might be some contamination.
If you are not getting any good colonies, you should confirm that you actually have plasmid DNA. You might also want to be sure you run a control for your PCR to be sure that negative results are real and not just PCR failures.
DH10B is good for larger plasmids.
I would say something wrong with your plasmid?
you are transforming the plasmid? where did you get the plasmid?
this plasmid must come from the E.coli.
Are you using the proper controls? For a positive control, use a plasmid that always works, and gets selected with the same antibiotic. Are you getting the same amount of colonies in the plate with no selection (without ampicillin)? What do you call "negative results"? Plasmid without the regions of interest? Maybe they have mutations in your gene. I would sequence the regions of interest with flanking primers.
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