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Help with plasmid extraction and redissolving?

plasmid extraction dissolving low yield

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5 replies to this topic

#1 molbiolnew

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Posted 22 May 2014 - 06:11 AM

Hi there.

I'm extracting MIGR1 plasmids using very standard techniques followed by Maxi Pred.

I end up with a nice big pellet of plasmid, at the very least 1ug/uL on nano drop (around 400-500uL volume)

Then I do phenol:chloroform:isoamyl based extraction and still get a large pellet at the end.

I'm having real trouble redissolving the pellet though. I'm trying to redissolve them in elution buffer from the Maxi kit and have left them overnight in the fridge with 500uL of the buffer, then tried shaking them at 20C in the morning, but I can still see the pellets and the nano drop suggests most of it has not dissolved (50ng/ul DNA in the soluble fraction now).

Can anyone suggest where the problem lies and what I can do to rescue the plasmids?

Thanks in advance.

 



#2 Ensyeh

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Posted 22 May 2014 - 08:12 AM

Hi,

I'm not sure , but resuspension of the pellet with TE^-1 ( 1X TE with one tenth EDTA)  and keeping it in 65C water bath may work. 



#3 molbiolnew

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Posted 22 May 2014 - 11:53 AM

Thanks for that. In the end, I did what you suggested, only at 37C (it was at hand). Worked a charm. Thanks for your help.



#4 bob1

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Posted 22 May 2014 - 12:29 PM

Note that dry DNA is relatively insoluble - overdrying after the ethanol wash steps is usually the cause of the DNA not dissolving easily.



#5 Trof

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Posted 22 May 2014 - 12:59 PM

bob1: I always wondered what to do if you overdry.. putting back some salts, 100% and then 70% ethanol step (as in generic precipitation protocol) and NOT overdrying it the next time should help, shouldn't it?


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#6 bob1

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Posted 22 May 2014 - 03:30 PM

I usually just put in TE and heat at 65 for up to 15  min.  Seems to work well.  I think salts would help too, but I usually don't want extraneous salts in my DNA.







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