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Transformation problem

transformation dh5a top10

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5 replies to this topic

#1 Christi

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Posted 17 February 2013 - 12:30 PM

Hello all, I am about to pull my hair out over this transformation so any advice would be much appreciated Posted Image

I am using Invitrogen's BLOCK-iT U6 RNAi vector entry kit, and have ligated my shRNA into the pENTR/U6 vector. I successfully transformed the One Shot TOP10 cells that came with the kit for one construct... problem is, now I have two more I want to do and we are out of the TOP10 cells that came with the kit. I have been trying DH5a and XL1-blue chemicompetent cells made in the lab with no luck. I know the cells are OK because the transformation with the positive control pUC19 worked splendidly. However, the LacZ shRNA that is in the pENTR/U6 vector does NOT work with DH5a or XL1-blue... and this is the same ligation reaction that worked with the TOP10 cells (frozen at -20, thawed once). To me, this suggests that there is problem with transformation of this vector into these cells types... but my PI thinks I'm just incompetent at transformation. The protocol I'm using is: 1-2 uL DNA into 50 uL competent cells, ice 30 min, heat shock 30-45 sec at 42 degrees, ice 5 minutes, add 250 mL LB (have also tried SOC), shake for 1 hr at 37 degrees, plate on pre-warmed kan plate O/N. Any ideas as to what I'm doing wrong, or why these competent cells wouldn't work for this vector? (and yes, I know the obvious solution is: buy more TOP10 cells! But money is tight and my PI thinks I should be able to get the other cell types to work.) Thanks!

#2 phage434

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Posted 17 February 2013 - 02:28 PM

Your competent cells are probably not very competent. You should measure, by transforming 10 pg of pUC19 or other similar plasmid. You should get 1000's of colonies if you have good cells. It is a mistake to think that if you can transform cells with 100 ng of vector that it is good for transformation.
See here to make good competent cells (and measure their competence):
http://openwetware.o...competent_cells

#3 Christi

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Posted 18 February 2013 - 03:27 PM

Your competent cells are probably not very competent. You should measure, by transforming 10 pg of pUC19 or other similar plasmid. You should get 1000's of colonies if you have good cells. It is a mistake to think that if you can transform cells with 100 ng of vector that it is good for transformation.
See here to make good competent cells (and measure their competence):
http://openwetware.o...competent_cells


Thanks phage434 for your reply! I think my cells are competent, because I tried transfecting with pUC19 and got a lot of colonies! I can also transfect with another plasmid, just not anything with the pENTR/U6 vector. Any other ideas?

#4 phage434

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Posted 18 February 2013 - 03:32 PM

It is not WHETHER you can transform with pUC19, but HOW EFFICIENT it is. How much DNA did you use in your control transformation?

#5 Christi

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Posted 18 February 2013 - 03:36 PM

Ah, I see your point. I transformed with 10 pg of pUC19 and got maybe several hundred colonies (hard to distinguish individual colonies, they were so close on the plate).

#6 phage434

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Posted 18 February 2013 - 04:17 PM

Then your cells are fine, and the problem is elsewhere.





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