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Sub-cloning and transformation


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17 replies to this topic

#16 och

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Posted 15 July 2012 - 03:57 PM

Sorry for replying late. I use QIAGEN II purification kit.
ok. I got you. Thanks so much.

#17 Nayeem991

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Posted 16 July 2012 - 10:34 AM

best of luck.... Posted Image

#18 jakester

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Posted 17 July 2012 - 11:17 AM

Do you make your own competent cells? If not, are you following the transformation protocol that comes with them? Two minutes seems like it is unnecessarily long for the heat shock. Reducing to 30-45 seconds should increase your efficiency 2-3x. (http://www.itescam.e...rsos/r72068.PDF) Also, every transformation protocol I have seen specifically says NOT to mix the plasmid and bacteria by pipetting up and down, since the competent cells are extremely fragile. A gentle flick or two will do fine. Finally, in my experience, increasing the amount of ligation product you use for transformation to 10uL as Nayeem suggests is likely to substantially reduce the transformation efficiency. I'd stick with 2uL.

I agree with Nayeem though, the first thing you need to do is run some controls, esp. with the uncut vector.




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