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low copy number plasmid purification kit

any suggestions ??

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4 replies to this topic

#1 nightingale

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Posted 12 September 2018 - 12:52 AM

Dear All, 

 

i am trying to purify my plasmid.

 

info. :

my plasmid is a low copy number.

my cells are XL 1 blue cells.

 

i used Hispeed Midi purification kit and i ended with a low yield.

 

from your experiences, if i want to end with approx. 1000ng/ul, which kit shall i use ??

 

Qiagen Hispeed Maxi ?? or do you prefer others ??

 

thank you!

 

always grateful,

nightingale 

 

 

 

 

 


" The more you learn, the more you realize how little you know ... "

#2 OldCloner

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Posted 12 September 2018 - 06:58 AM

Hi Nightingale,

 

Our lab used Qiagen kits, either the QIAfilter maxi or the Large Construct kit (which is for plasmids or BACs that are both large and low-copy [LC]).  Most kits should work but you have to follow the directions for LC plasmids, and that usually means larger culture volumes. Did your kit have special instructions for LC plasmids and did you follow them?

 

Obviously you will get lower yield than you are used to with high copy plasmids.  If the quality of the prep is good, just low yield, you can make several preps and pool them, then concentrate them by ethanol precipitation to get the desired concentration. Or, stepping up from midi to maxi should help.



#3 nightingale

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Posted 12 September 2018 - 10:59 PM

thank you OldCloner! 

 

yes i followed the LC instructions, but still i got low yields.

But, the kit already stated that with LC plasmids expect considerably low yields!

 

i did so, but ended having much to deal with, so i thought if i scale up to the maxi, things will be easier,

even if i pooled, at least each single yield would be higher than my previous.

 

thank you again!


" The more you learn, the more you realize how little you know ... "

#4 Trof

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Posted 19 September 2018 - 06:50 AM

Yeah, maxiprep for low-copy plasmid purification with required 1ug/ul concentrations would probably be a better choice.


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#5 Curtis

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Excellent

Posted 16 October 2018 - 10:25 PM

Last time, after failing to grow with chloramphenicol, we used a reagent by Epicenter. I dont remember its name but it really helped us. We also used 300+ rpm and longer incubation.





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