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Plasmid size problems!


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3 replies to this topic

#1 Kami23

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Posted 17 June 2010 - 05:58 AM

Hey all,

Im having a problem stuffing a rather large (14kb) plasmid into my competant E.coli. The concentration of the plasmid is around about 500ng/ul and im using 2 to 5 ul per 50ul of cells. The cells are ultracompetent XL10 Gold cells. I dont use B-ME but maybe i should. Does anyone have any suggestions as to how to solve this problem?

B

#2 rkay447

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Posted 18 June 2010 - 07:39 AM

I have just started working with a rather large plasmid (13kb) for the first time and have had a horrible time getting a decent DNA prep as well. I can only get about 400ng/ul so I did two midi-preps and ethanol precipitated the DNA. I then resuspended the entire pellet in a very small volume of water and managed to get a very clean prep around 1.5ug/ul. I don't think you'll be able to fix the problem by changing the cells you are using and your best option is to do what I did and precipitate the DNA from a large purification and resuspend in a small volume.

#3 phage434

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Posted 18 June 2010 - 07:46 AM

Why do you think you need such high dna concentrations? 10 ng should give hundreds or thousands of transformants with decently competent cells.

#4 mahsa

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Posted 25 June 2010 - 10:27 AM

I quote Current protocols, essential lab techniques:"In a suspension of competent cells, only a small proportion are actually competent. For that reason, the efficiency of transformation decreases as more DNA is added (i.e., fewer transformed bacteria
will be obtained per microgram plasmid added). A quantity amounting to 1 or 2 ng plasmid is usually
sufficient. It is important, however, to minimize the volume of the introduced DNA, to avoid overly
diluting the competent cells. A 1:5 ratio of DNA to cells gives good results."

so, you may wanna double check the plasmid amount you add to the transformation reaction.
good luck




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