Now, I'm in the step of plasmid construction in E. coli.
But I found a big problem because of an undesired or contaminated plasmid DNA
(some one called Ghost plasmid) in my transformants (more than 40%).
I already test my E. coli competent cell. The result showed that no colony in negative control.
So, I think maybe a contaminated plasmid come from a container that use for staining gel by Ethidium bromide, or container that use for destain EtBr, or in a plastic tray that use with electorphoresis machine.
Could anyone suggest a solution or chemical (should safe and simple) that use for washing and remove a contaminated plasmid DNA from my container, and a plastic tray in electorphoresis machine?
How did you confirm presence of the contamination in your plasmid DNA sample? Your negative control is not giving any band. Also, what type of transformation protocol are you using, electroporation or chemical?
If you know the plasmid size of your interest and it is different from the contaminant plasmid size, then cut the DNA band out and run DNA extraction using the extraction kit from promega, Wizard® SV Gel and PCR Clean-Up System.
Hi, if you want to wash your electrophoresis apparatus I usually use 1M NaOH, at least 1h. Rinse the chambers and put inside also your comb and the gel tray. After this wash very well with distilled wate, and if possible with DEPC or MilliQ H2O.
Good luck
Dear Singh,
I found that a lot of my transformants contain some plasmid that the size of plasmid is not correct, and I never use a plasmid that have a size like that. And I use rubidium chloride and heat shock method.
Dear Tiong, and Arcangelis
Thank you for your suggestion.
Hi Tatpong
as Hung King Tiong suggested you, you should purify your plasmid if the digested products are different in lenght. Otherwise you should consider if you are having recombination between plasmid and genomic DNA, due for example to insertion elements in your costruct.
best
silvia
Hey Tatpong,
if, as Silvia put it, you have some recombination, I would suggest you check your transformants by colony PCR in the region of your insertion, then you will more or less quickly find transformants harboring your desired plasmid. If those have an additional plasmid (your "ghost plasmid"), just isolate the plasmid-DNA and make a second transformation round with several dilutions, to reduce the probability of double-transformation.
Best regards,
Christian
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