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Maxiprep problem

Maxiprep problem - DNA Extraction Forum

Maxiprep problem - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


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  #1  
Old 11-28-2008, 09:44 AM
Pipette Filler
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Question Maxiprep problem



I have for a while experienced very little or no yeild with maxipreps of my plasmids (vector + insert = 10 kb). At the moment I use Sigmas maxiprep kit (but I have also tried kits from Jet star and Qiagen). However I dont think it is the kit that is the problem. I always pick fresh colonys in 2ml LB + Amp(always newly autoclaved solutions and fresh amp) incubate over night (37oC, shaking)and perform minipreps on. The minipreps always have good yield and restriction cleavage shows I have the correct product.

The problems arise after this step: I incubate (same conditions as above) LB, amp and a little from the miniculture (1/1000 of the total vol) over night. I control that OD at 600 nm is the desired one and then perform maxipreps. Since I didnt have any maxi yield last time I also performed a miniprep on I.5 ml of the maxiculture showing also no product, indicating that something must have happened during the incubation of the maxi culture, but I just cant figure out what is going wrong?
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Old 12-15-2009, 06:47 PM
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Default Re: Maxiprep problem

Is the maxiprep draining quickly? If your bacterial volume is too high, it can clog the pores. If this is happening, try using twice the volumes of lysis, resuspension, etc.
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Old 09-30-2010, 12:27 AM
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Default Re: Maxiprep problem

After the lysis spin and then also try using a filter to obtain the supernatant. This will remove most the cell gunk before you add the supernatant to the column. Also most kits are only good for about 2 years, the columns might be bad. Another thing, make sure you are properly equilibrating the columns before use.

~knighbri
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