3 replies to this topic

[dygibbs]

I'm having a hard time trying to obtain adequate yields (average: .150 ug/ml) on maxipreps:  a 6.5kb construct (2.7kb insert cloned into a 3.8 kb vector).  Any suggestions?

[vgupta]

incomplete lysis could be the cause.  Double the amount of lysis solutions.

[Victor]

Try the Endofree maxi kit from Quiagen.  You may also want to look at the protocol insert in the kit.   It is very informative.  

I've only done three preps.  My last 2 yields were in the 250 microgram range.  My first prep was 600 micrograms!  The wavelength scans were very clean, A260/280 ratios were excellent, and the diagonistic cuts were perfect.  Also, the endofree kit improves transfection efficiency.

Make sure your bacteria are growing well.  You should have about 1 to 1.5 gram of pellet for lysis.  Also, try reading OD 600 to see how well your bacteria are growing.   I haven't done the OD reading but I did check the pellet mass.

Cell lysis is probably also an important factor.  The endofree kit is a bit tricky at the lysis step.  Make sure most of the proteins/lipids float up to the top of the column and that your column solution runs clear when you apply the plunger.

[judep]

Try using superbroth instead of LB- I use 400ml and then double the amount of all solutions (20ml instead of 10) and it always works!!