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Problems with plasmid DNA extraction


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#1 Lfs

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Posted 14 July 2013 - 12:30 PM

Hi guys!

Pleeease, I need your help! Posted Image

I'm working with pEGFP-C3, pHM6-HA and pDEST-V5 plasmids (with the inserts) in order to use them in cell culture. Theoretically, they are high copy plasmids. Growing 1 single colony in 5 ml of LB media should be enough. But it is not! The last time I use a mini kit (Sigma) for extraction, I got less than 50 ng/ul (20ul) of a impure DNA. Oddly, it happens only with these 3 plasmids. I try the same kit with my pTRG plasmids, and it worked fine (more than 300ng/ul).

Well, I use XL1-blue competent cells for subcloning, grow them in 5 ml (+ atb) at 37*C overnight and extract de DNA. It should be simple! Posted Image And I normally do the toothpick minipreparation in order to identify bacterial colonies containing the recombinant plasmid (molecular cloning manual).

I already tried doing a Midi prep with alkaline lysis (without kit) and the plasmid's amplification using chloramphenicol... and nothing!

Do you guys think that my plasmids might be toxic for the cells? Should I grow them in 30*C? Any suggestions?Posted Image

Thank you!!

Edited by lschier, 14 July 2013 - 12:30 PM.


#2 Ahrenhase

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Posted 14 July 2013 - 01:16 PM

I don't quite understand the use of chloramphenicol since these appear to be kan and amp resistant plasmids, but I will trust you know what you're doing.

I had a similar problem which was solved by growing at a lower temperature. In my case, the cultures would never grow to turbidity at 37C.

#3 Lfs

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Posted 14 July 2013 - 05:14 PM

I read that plasmid DNA yields can be improved by adding chloramphenicol (170ug/ml) to the culture medium (Molecular Cloning, Sambrook and Russel), but in this case it didn't work.

I'll try lower temperatures! Thank you!

#4 Lfs

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Posted 05 August 2013 - 04:14 PM

Hi there!

 

Lower temperature didn't work! unsure.png 

 

Actually, the culture growth was fine but after doing a midiprep, I got only 200ng/ul of plasmids (with RNA contamination)! blink.png I need, at least, 500ng/ul for a transfection protocol. By the way, can I use DNA simple with RNA contamination for transfecting mammalian cells?

 

Do you, guys, think that the problem is the XL1-blue? Should I try another strain, like DH5 alpha? Should I try something else? 

 

Any suggestions? blink.png

 

Thank you very much! rolleyes.gif






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