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A problem for miniprep

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A problem for miniprep

Postby bluetutu » Sun Mar 13, 2011 7:15 pm

Hello Everybody^^,
Very nice to find this forum^^. I have a question about my recent work. I did some cloning work these days, and Yesterday, I did miniprep from a BL21 culture, but it seemed contaminated by bacterial genome (complete white on the gel, I changed fresh TEA buffer when doing the Electrophoresis, and loaded untraited plasmid as control :cry: ).This is the first time I use BL21 expression E. coli system. I did culture in LB buffer containing 1% glucose for repressing the toxic protein expression. I did miniprep with Qiagen kit, quite good concentration measured by Nanodrop, around 400 ng/ul, 260/280 is 1.9 (no protein contamination). The problem occurred when I used restriction enzyme for digestion...No specific band and even for uncut plasmids....I did twice, both failed....The incubation time of culture was equal or less than 16h...I just suspect that if glucose has chance to affect the miniprep???? :(


Waiting for answer^^
Have a nice Night for everbody^^ :D

Greetings
bluetutu
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Re: A problem for miniprep

Postby protold » Mon Mar 14, 2011 7:23 am

Hola, glucose only increases the OD (mass) of the culture, but 0.5 g/l would be enought. But before transformation in BL21 you have to obtain the plasmid in a cloning strain as DH5a or XL1blue or similar. It´s better characterize the plasmid in these strain than expression ones, and after well characterized it you have to trasform expression strains
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Postby bluetutu » Mon Mar 14, 2011 2:07 pm

Thanks a lot. I will try XL1 blue, DH5a I tried twice for transformation, but both failed. XL1blue might be a good way...God bless me...


Thanks again^^
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