I'm trying to extract my constructed plasmid from E.Coli Top 10 cells for which colony PCR is positive and failed multiple times. Plasmid is pET-28a. I'm using Tiangen Mini Plasmid kit and the provided protocol for plasmid extraction. Any help would be appreciated. Thanks!
A lot of little things can go wrong with a miniprep. But with a high copy plasmid like pET-28a, as long as you're using the correct buffers, you should still have some DNA at the end. How are you preparing your starter culture?
Stephen Cohen, Thanks for your prompt reply. Well, I pick the positive colony from the plate and culture in broth. With the same kit, i'm extracting the wild type plasmid successfully. Stephen Philip Cohen
Stephen Philip Cohen Yeah I've used the fresh antibiotic solution in broth. I repeated the experiment 3 times and got the same result.
Does the overnight culture grow fast? Is it very turbid? For low copy plasmids, to increase mini-prep yields, I will culture in 5 mL of LB for 24 hours.
Have you tried running PCR on the overnight liquid culture to confirm that the plasmid is still in the culture before mini-prep? You can dilute 1 uL of cell suspension in 50 uL of water, then use that as template in your colony PCR protocol.
I recommend you ensure the lysis step is conducted quickly, because lysis solution can damage DNA. In the instructions for the Tiangen kit, it recommends a maximum of 5 minutes in lysis solution (step 4). I recommend doing this step quickly, e.g. within 10 seconds, or as soon as the bacterial suspension turns clear.
Hello
Currently I have used this plasmid. After having the recombinant plasmids (it proved with PCR colony) I used 3 solutions and at the end I washed with ethanol 100 % and 70% And the pellet resuspending at TE1X. After I used Invitrogen kit and I didn't have problems but I remember that I have poor concentration. Other great alternative is after the solution III putted it all in the column and continue with the purification according to the protocol of kit without ethanol. If you want I can send you my own protocol.
On other side, I think the Buffer binding could be the problem, maybe your plasmid is eluting in the collection tube. I recommend you proving this problem: in a agarose gel running the first elution of the column.
Keep in touch. Good luck!!
Cleidy
Stephen Philip Cohen yeah, I've been doing all this according to your suggestions but same results. May be I need to change the plasmid.
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