Hi
I have a problem in my transformation part of project
after preparation of competent cells and transformation nothing growth in plates
protocol of competent cell is this
5ml of renewed bacteria in LB broth in certain OD
centrifuge 3000rpm 3min - remove liquid part
add 1ml of CaCl2 and re suspend .incubate 30 mins on ice
centrifuge 3000rpm 3min - remove liquid part
add 670 MicL CaCl2 and re suspend . incubate 30 mins on ice
centrifuge 3000rpm 3min - remove liquid part
add 300 MicL of CaCl2
incubate on ice 30mins
heat shock 45sec in 42`C
immediately transfer tube on ice 2mins
add 900 MicL LB medium
incubate 4 hours in falcon tube in 37`C shaking 190 rpm
then culture in this medium :
LB Agar + Kana(50Micg/Ml) + tetra (10Micg/Ml)+ 100micg/ML X-Gal)+7micg/Ml gentamycin + 40 micg/ml IPTG
but nothing growth after 48hour in 37`C
what is your advise or where is the problem
Hi Amir,
Firstly, for making competent cells, one of most critical parameter is the right O.D at 600 nm., It is generally recommended to be between 0.2 and 0.5. Ideally the mid log phase cells OD 0.5 works just best.. I would like to tell you that there is a competency buffer (CCMB80) for preparing DH10Bac and as a good practice I would advice you to stick to that buffer. You may find the details online.
But, why did you heat shock the cells without adding any DNA, I hope your intention was to transform these prepared competent cells to test their Transposition efficiency!! For transformation with donor construct, use 100ng DNA..
Secondly, It is important that you confirm the selection marker (Gentamicin or Ampicillin) on your transfer vector., and supplement the drug along with the culture medium when you prepare plate.
Hope this Helps!
Good Luck :)
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