Hello.
I have been doing Maxipreps with plasmids in a pUC19 vector and they worked great! However, when I try to do a plasmid prep for another plasmid, they are failing. I am not getting ANY DNA.
I am using the same protocol - streak on plate, pick a colony, grow in 5 ml LB, then inoculate 250 ml of 2YT with 2.5ml of the starter culture. I grew this overnight at 37 degrees and 200 rpm.
My GOI is in a pET30 backbone and includes a 10xHis, 3xFLAG and TEV site.
I should also note, that when I did a mini prep, it worked (however, it was not supercoiled plasmid, this might be due to old columns)
ANY recommendations would be greatly appreciated. Thank you.
I think your getting over saturated cultures especially since you are using 2xYT. This will lead to clogging of columns. Dilute 1:1000 and not 1:100 and grow at 30C. It could be that your cells grew more slowly with pUC19 since it has a much higher copy number than pet30 and hence were not as saturated. For exact O.D harvest the cells as indicated in your kit's manual and if you keep getting problems you might want to switch to LB instead.
P.S make sure you are harvesting from a delta endA strain otherwise you might get plasmid degradation unless you wash with an isopropanol containing buffer.
I would suggest isolating it manually (not kits) and without using columns ... I was facing a similar problem and solved it when I left the columns aside.
Hanna Alalam I will adjust it to what you recommend and see how it goes. Thank you so much.
I have used the similar vector (pET30a-LIC) and it worked perfectly producing a yield usually over 0.7mg of DNA from 250mL culture. If you won’t mine will you try 6 hours instead of the overnight? I see that you grew them overnight at 37 degrees and 200 rpm. Everything you did so far meant that it should work at least. Can you also please tell us the type of competent cells you used? Was it NovaBlue, or BL21? Thank you and have a great time in experimentation.
Are you having issues with any other kanamycin selection vectors? Check the date/concentration/storage of your kanamycin. If the antibiotic selection isn't consistent then your bacteria could lose the plasmid.
One thing you could try is a miniprep from an aliquot of your large culture. That would help determine if it's a plasmid/antibiotic issue or a kit issue.
Basically, if you can miniprep up the plasmid from your large culture then it's a kit issue leading to the lack of plasmid isolation from your maxi kit. If you can't, then it's something else like the antibiotic.
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