What is the best method, condition, and strain of E. coli that can be used during transport and storage of large size plasmids "27~35 kbp. Although, I was able successfully transport the target plasmid, 27.3 Kbp, into E. coli JM109 using my preparation of an electro-competent cell and Bio-Rad electroporation protocol, and I confirmed the success of transformation using PCR check and plasmid size after extracted from the positive colony "5ml", but that positive colony didn't show good keeping for the plasmid during the 2nd propagation and has generated smaller plasmids 8 and 4 kbp with diminish its production of the correct plasmid gradually until almost disappeared.. Is it possible to non-fully transcript the whole plasmid during reproduction???
I also tried with the heat shock methods using both E. coli JM 109 and NEB 10 beta strain and many colonies grown on LB-Ampicillin plate; however none of them generates the correct size of the plasmid "27kbp".
I wouldn't use JM109, it has the F plasmid integrated into its chromosome and that could possibly cause some problems if the F plasmid proteins interact with your plasmid in any way.
Also, how are you checking the size exactly? >20 kb is often unresolveable on a standard agarose gel.
Then, I would also check to make sure it's not just that your ampicillin or miniprep reagents went bad.
Can you give us more info about the plasmid?
Thanks Alexandra Johnson for valuable information about F plasmid.
I confirmed the plasmid size by two ways (1) single, double digestions. (2) PCR for each cassette that had inserted and also for the plasmid it self and compared with two different DNA ladder
https://www.neb.com/products/n3239-quick-load-1kb-extend-dna-ladder#Product%20Information and
https://www.genedirex.com/1kb-dna-ladder-rtu-ready-to-use/
For reagents, it's no problem where I am using QIAGEN kit and used well for other size of plasmids and also used well to obtain the first generation of the big plasmid.
I have no problem with JM 109 at all during transform 4,6,8,10,12,14,17 kbp plasmids. This problem appeared only after insert another 10 kbs of DNA to previous 17 kbp plasmid.
Regards,
Thanks Ali Mahmoudpour
Previously, I commonly used DH5 alpha and it will be my next destination, but before that, i would like to ask you about what did you use during transformation? heat or electroporation methods???
I am confirming your observation of Undigested plasmids have unpredictable mobility with the degree of supercoiling, relaxing, or in mode of replication forming interlinked dimers.
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