Hi every one. I am facing problems in the yield of Plasmid DNA isolation. I need your suggestions:
I am using high copy number plasmids like pTT5, pcDNA.
I am using MDI Mega and Giga kits for plasmid isolation following the manufacturers manual. But I am not getting the yield as per the manual. It is supposed to be 15-20 mg but I am getting 8-10 mg and the yield is consistent for about 150 different samples (8-10 mg only).
I used Carbenicillin instead of ampicillin, No use
I increased the shaker rpm from 180-200, then also the same result.
Most importantly I am using circle grow instead of LB broth.
Can any one guide me in this problem. Looking forward for your reply.
Thank You.
Try to grow bacteria in 4 ml of LB. I guess, penaultimate step of plasmid extraction is adding of ethanol. Here, perform additional centrifugation at 10000 rpm/4 min. This will help to eliminate all the ethanol from the column. Then, add 50 µl of pre-heated (72ºC) elution buffer (TE). Incubate at RT for 3-5 mins. Centrifuge for 2 min. Now, take the elutent and add to the column again. Incubate at RT for 3-5 mins. Centrifuge for 2 min.
You can increase the amount of inoculum. I tried 10 mL of cell and separated them into 2 tubes. At the very last elution step, you combine both columns and elute them into a single tube. Other than, you can increase the incubation time when DNA is binding to the column.
try Invitrogen PureLink kits, available up to Giga scale - standard and endo-free versions. consistently high yields. tricks how to boost plasmid yield: heat elution buffer to 37-50C - this gives at least 20% increase. depending on the resin- double elution or larger elution vol helps.
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