pGEX 2T vector maxi prep or mini prep

[david1259]

I need to seqeunce my two DNA in pGEX-2t vector in BL21 cells.
I tried to mini prep and Maxiprep however the DNA conc is too low to sequence.
Is there any way I can increase DNA amount?


I usually use 1ml culture for miniprep or 50ml culture for maxiprep.

Please, help me.

[kmunson779]

1) I would not suggest using BL21 cells to propagate plasmids. They're not as good as DH5a (for example) at avoiding DNA recombination and mutation issues.

2) I use 5 mL of culture for a miniprep and 50 for a midiprep -- for high copy plasmids, the Qiagen maxi kits take 100 mL of culture. You could try scaling up.

3) If it's simply a concentration issue rather than yield, an ethanol precipitation step will give you higher concentration plasmid.

[Beth]

A related question...how well does the pGEX plasmids prep up? Compared to other plasmids miniprepped along side the yield of these are extremely low....only about 60ng/ul and was wondering if that was due to the nature of these plasmids?