Transformation, no colonies! - (Jul/10/2008 )
QUOTE (ph3no @ Jul 11 2008, 12:15 PM)
QUOTE (biorules @ Jul 11 2008, 09:30 PM)
HI THERE! I had my own cloning blues for months until today,, when I got my correct colonies.. 
I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... 
Good luck! 
I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... Good luck! good to hear,
I was thinking that 90 seconds at 42 degrees is far too long, use maximum a minute, I use 40 seconds or less...
Then, when using ampicillin, there is no need incubating in LB without antibiotic, you can directly plate the cells after they have been 3 minutes on ice, maybe bring them to room temperature for a few minutes prior to plating.
and get rid of the spinning and resuspension as well...
I agree with ph3no...90 seconds seem too long...I do 30-35 seconds at 42C...also after 2min of ice after that, I sometimes add 300ul of pre-warmed SOC and keep it in the shaker at 37C for 45 min. I then plate the whole of it on to LB Amp plates...
-biorules-
QUOTE (biorules @ Jul 11 2008, 11:37 AM)
QUOTE (ph3no @ Jul 11 2008, 12:15 PM)
QUOTE (biorules @ Jul 11 2008, 09:30 PM)
HI THERE! I had my own cloning blues for months until today,, when I got my correct colonies.. I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... Good luck!
I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... Good luck! good to hear,
I was thinking that 90 seconds at 42 degrees is far too long, use maximum a minute, I use 40 seconds or less...
Then, when using ampicillin, there is no need incubating in LB without antibiotic, you can directly plate the cells after they have been 3 minutes on ice, maybe bring them to room temperature for a few minutes prior to plating.
and get rid of the spinning and resuspension as well...
I agree with ph3no...90 seconds seem too long...I do 30-35 seconds at 42C...also after 2min of ice after that, I sometimes add 300ul of pre-warmed SOC and keep it in the shaker at 37C for 45 min. I then plate the whole of it on to LB Amp plates...
Thanks for the suggestions!! I am going to give some of them a try this weekend and will post later as to what works/doesnt work incase anyone else is having similar problems!
-SMB1980-
QUOTE (SMB1980 @ Jul 11 2008, 02:42 PM)
QUOTE (biorules @ Jul 11 2008, 11:37 AM)
QUOTE (ph3no @ Jul 11 2008, 12:15 PM)
QUOTE (biorules @ Jul 11 2008, 09:30 PM)
HI THERE! I had my own cloning blues for months until today,, when I got my correct colonies.. I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... Good luck!
I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... Good luck! good to hear,
I was thinking that 90 seconds at 42 degrees is far too long, use maximum a minute, I use 40 seconds or less...
Then, when using ampicillin, there is no need incubating in LB without antibiotic, you can directly plate the cells after they have been 3 minutes on ice, maybe bring them to room temperature for a few minutes prior to plating.
and get rid of the spinning and resuspension as well...
I agree with ph3no...90 seconds seem too long...I do 30-35 seconds at 42C...also after 2min of ice after that, I sometimes add 300ul of pre-warmed SOC and keep it in the shaker at 37C for 45 min. I then plate the whole of it on to LB Amp plates...
I've still got nothing, I transformed with 50ng of my original plasmid and STILL have no colonies!! I am beyond frustrated at this point!!! I've never had a problem where NO colonies grow!
Thanks for the suggestions!! I am going to give some of them a try this weekend and will post later as to what works/doesnt work incase anyone else is having similar problems!
-SMB1980-