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#1 D.Luffy

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Posted 24 April 2018 - 07:27 AM

After setting up ligation,I proceeded with transformation. Our lab usually does,5ul ligation mix to 50ul competent cell(mach1 we are using). Unknowingly,I transferred 10ul ligation mix to 50ul com cells,from that 5ul I stored at -20C(for back up) and the rest I did as per normal transformation protocol and plated it. Do colonies grow? And mainly,when using the stored 5ul for new transformation will it work? Help me please!!

#2 bob1

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Posted 24 April 2018 - 08:32 AM

Depends on the protocol, but 10 ul ligation in 50 ul of cells is probably too much. Ideally you want 5% or less ligation mix in your cells. Having said that, I would be surprised if you didn't get at least some colonies.

 

The cells stored at -20 will probably not work once thawed.

 

If you have some ligation stored, that should work, even if it is less than 1 ul.



#3 D.Luffy

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Posted 27 April 2018 - 09:55 AM

Yeah, I got colonies(around 40-50). On screening it was not the expected one!!
And for transformation of that stored in-20, if I directly go to heat shock and continue the protocol? Any chance?

#4 OldCloner

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Posted 12 September 2018 - 07:45 AM

So, you froze your back up cells BEFORE finishing the transformation? Their competent properties would be lost. You'd make better use of your time re-doing the experiment with a fresh aliquot of comp cells.. You can re-transform any ligation that you stored frozen (or probably refrigerated, 1 night). I agree with bob1 about the volume to put in-less is more in this case.

 

You can store transformed cells to plate later by adding 15% sterile glycerol and freezing at -20 C.  You can even just put them in the fridge overnight (no glycerol needed) until you see the results from the previous day's transformation, but for longer storage, freeze them. There will be a slight loss of efficiency, but no big deal.






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