Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

transformation efficiency decreased


  • Please log in to reply
7 replies to this topic

#1 moljul

moljul

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 127 posts
2
Neutral

Posted 04 August 2009 - 01:06 AM

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

#2 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 04 August 2009 - 02:52 AM

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

#3 moljul

moljul

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 127 posts
2
Neutral

Posted 04 August 2009 - 03:10 AM

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.

#4 Qundo12

Qundo12

    E. coli farmer

  • Active Members
  • PipPipPipPipPip
  • 88 posts
2
Neutral

Posted 04 August 2009 - 10:13 PM

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 ;)

#5 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 04 August 2009 - 11:06 PM

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.

#6 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 04 August 2009 - 11:10 PM

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.

#7 Qundo12

Qundo12

    E. coli farmer

  • Active Members
  • PipPipPipPipPip
  • 88 posts
2
Neutral

Posted 05 August 2009 - 01:23 AM

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 ^^

#8 little mouse

little mouse

    Missele, the little mouse

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 172 posts
2
Neutral

Posted 05 August 2009 - 03:11 AM

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 ;)




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.