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transformation efficiency decreased - storage conditions of competent cells (Aug/04/2009 )

hello,

i have a question to the transformation efficiency of my competent E.coli:

last week we defrosted our -80C freezer, a colleague of mine put my competent cells into the -20C feezer during this defrosting-process! i checked it after a while and put the E.coli again -80C.

could this (-80C) - (-20C) - (-80C) temperature changes could harm my cells, because since this timepoint my transformations didnt work anymore. i have to check every point of cloning/transformation to be totally sure, before i order some new E-coli.

thanks for suggestions

-moljul-

try to transform your bacteria with 50 ng of cirular DNA. You will see if your bacteria are still competent or not.

-little mouse-

little mouse on Aug 4 2009, 03:52 AM said:

try to transform your bacteria with 50 ng of cirular DNA. You will see if your bacteria are still competent or not.


thanks.
ive already worked with circular DNA (puc19) and got less transformants than before.

-moljul-

I routinely thaw-freeze my competent cells from -70 deg -> 4 deg (on ice) -> -70 deg and use each tube of cell about 3-4 times but the transformation is still good. Which method you used for transformation? btw, why do you have to order new E. coli? just prepare the competent cell again, it's fast ;)

-Quasimondo-

Quasimondo on Aug 5 2009, 08:13 AM said:

I routinely thaw-freeze my competent cells from -70 deg -> 4 deg (on ice) -> -70 deg and use each tube of cell about 3-4 times but the transformation is still good. Which method you used for transformation? btw, why do you have to order new E. coli? just prepare the competent cell again, it's fast ;)


your transformations are still good ?! is it electrical or chemical competent cells? Do you have still good transformation of ligation products?
If yes I would like to know your protocol.

-little mouse-

moljul on Aug 4 2009, 01:10 PM said:

little mouse on Aug 4 2009, 03:52 AM said:

try to transform your bacteria with 50 ng of cirular DNA. You will see if your bacteria are still competent or not.


thanks.
ive already worked with circular DNA (puc19) and got less transformants than before.



If I were you, I would keep these bacteria for circular DNA and order new competent bacteria.
I never tried by myself to put the bacteria to 4C and then back to -70C, but I was told not to do.
Maybe your bacteria stood outside the freezer too long, and I put a bet that nobody mixed again the bacteria with the glycerol before to freeze them again.

-little mouse-

I use electroporation with 0.2 cm gap cuvette, Bio-rad device, 2.5kV, 4ms. All of competent cell, cuvette are kept on ice. Mix about 100ug DNA (2-3ul) with 40-50ul competent cell, transfer to cuvette then pulse. Immediately recover the cell by 1ml LB, then inoculate at 37 degree for 1 hour in the shaker. Centrifuge, discard 900ul, resuspend the cell in the remaining 100ul, then spread on antibiotic containing plate.
I prepare competent cell by myself every 1-2 month. I use XL1-Blue strain: Grow the cell in 100ml LB until it reaches OD600 about 0.9 or 1. Keep the culture on ice for 30 min to stop growth. Centrifuge at 4 degree for 15 min. Wash twice with sterilized water (cold) in 40ml, then once with 10% glycerol in 25ml. Resuspend cells in 300ul 10% glycerol, then keep at -70 degree. It's all ^^

-Quasimondo-

Quasimondo on Aug 5 2009, 11:23 AM said:

I use electroporation with 0.2 cm gap cuvette, Bio-rad device, 2.5kV, 4ms. All of competent cell, cuvette are kept on ice. Mix about 100ug DNA (2-3ul) with 40-50ul competent cell, transfer to cuvette then pulse. Immediately recover the cell by 1ml LB, then inoculate at 37 degree for 1 hour in the shaker. Centrifuge, discard 900ul, resuspend the cell in the remaining 100ul, then spread on antibiotic containing plate.
I prepare competent cell by myself every 1-2 month. I use XL1-Blue strain: Grow the cell in 100ml LB until it reaches OD600 about 0.9 or 1. Keep the culture on ice for 30 min to stop growth. Centrifuge at 4 degree for 15 min. Wash twice with sterilized water (cold) in 40ml, then once with 10% glycerol in 25ml. Resuspend cells in 300ul 10% glycerol, then keep at -70 degree. It's all ^^



Thank you for your protocol ;)

-little mouse-