I am working with expression plasmid (2.6 kb) and I ligate it with an insert of 15.186 kb to it. I grew my positive colony (identified by colony PCR) on LB (6 ml+ selective antibiotic overnight and I purified my plasmid using Qiagen mini-prep and I got 20-30 ng/ul which is very low as I need 1 ug of this plasmid for my transfection. By the way, I did some tricks to increase the concentration such as using 30 ul instead 50 ul of pre-warmed EB elution buffer and I doubled the volume used from lysis buffer, buffer 1 and buffer N3 and I got approximately the same range of concentration 20-30 ng/ul. Are there some soultions to increase the concentration from 20 to 200 ng/ul?
The main issue is that you're only starting with a 6 mL culture. Grow a 30 ml culture and prepare the culture in batches of 5 mL (or more!). Load the lysates sequentially onto the same column to increase your plasmid concentration.
As long as you ensure full lysis you may be able to use pellets from a larger volume of culture than the recommended 5 mL. A helpful tip here is that once you have pelleted the cells, remove MOST of the supernatant leaving about 10 uL on top of the pellet. BEFORE you add P1, vortex the tube to completely loosen the pellet making sure there are no 'lumps' of cells. It should end up as a smear across the inside of the tube. Only then add P1 and vortex again. The cells will then be fully dispersed ensuring complete lysis of the cells upon addition of the lysis buffer.
I agree with the comments given by Marcus. If you are not still getting the desired amount by applying Marcus's recommendations, you can also try a Midiprep kit to purify your plasmid from large volume cultures (from 50-100ml overnight cultures). Good luck!
Hello Abdou,
I'd like to add a few tips on the low-copy plasmid isolation to the top of the very good recommendations from comments above.
When I want to isolate my low-copy plasmid, I prepare 20ml overnight culture and spin it down into 4 tubes (5 ml of culture per tube). Then I continue according to the protocol provided by kit manufacturer, except that after adding the neutralisation buffer I centrifuge samples for 5 min at 4 ºC. It will ensure that the precipitate stays stuck on the tube's wall very strongly and you can take all the supernatant very easily. Regarding elution, I use 50ºC water and in addition to that I put the first eluate back to the column and perform second elution.
With this procedure I usually obtain 200–350 ng/µl.
I hope I helped a little.
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