Maxiprep problems - (Jun/17/2008 )
I need to do 10 maxipreps on 10 plasmids (Amp resistence).
First, I did transformation with the one shot top 10 competent E.coli (that's the only competent our lab has). There were a lot of colonies on the Amp plates. I carefully pick single colony from each plate and transferred it to 2ml Amp LB media. But only 4 out of 10 grew in the LB media. I continued to transfer 100ul bacteria to 150ml Amp LB media, and used Sigma-Aldrich HP Maxiprep kit to do maxiprep. But after adding resuspending and lysis buffer, 2 of them were not cleared. It looks like the cells were not lysed. The other 2 appeared normal at this stage, but finaly only 1 get plasmid, the other one get nothing.
What could be the problem?
Did you pick the right colonies? Satelite colonies are much smaller colonies compared to the right colonies.
How long incubate the inoculated culture? What was the incubation temperature? If you incubate at low 20C, you don't have enough cells to do mini-prep or maxi prep.
good luck!
First, I did transformation with the one shot top 10 competent E.coli (that's the only competent our lab has). There were a lot of colonies on the Amp plates. I carefully pick single colony from each plate and transferred it to 2ml Amp LB media. But only 4 out of 10 grew in the LB media. I continued to transfer 100ul bacteria to 150ml Amp LB media, and used Sigma-Aldrich HP Maxiprep kit to do maxiprep. But after adding resuspending and lysis buffer, 2 of them were not cleared. It looks like the cells were not lysed. The other 2 appeared normal at this stage, but finaly only 1 get plasmid, the other one get nothing.
What could be the problem?
If your cells are not lysing then chances are that you didn't grow e.coli. I would check your cells before you redo the transformation to make sure they are not contaminated, also check the LB that you use to do your tranformation as well. Try doing a miniprep before growing the larger prep to save yourself the time of growing things that aren't e.coli (you may not get much DNA, but if your cells are lysing properly then at least you have a chance). good luck.
Those one shot top 10 cells are in the original stock of invitrogen box. I did aliquot 10ul of cells into each tube to do the transformation. This time, I made fresh Amp plates, and redid the transformation. Wait to see.....
First, I did transformation with the one shot top 10 competent E.coli (that's the only competent our lab has). There were a lot of colonies on the Amp plates. I carefully pick single colony from each plate and transferred it to 2ml Amp LB media. But only 4 out of 10 grew in the LB media. I continued to transfer 100ul bacteria to 150ml Amp LB media, and used Sigma-Aldrich HP Maxiprep kit to do maxiprep. But after adding resuspending and lysis buffer, 2 of them were not cleared. It looks like the cells were not lysed. The other 2 appeared normal at this stage, but finaly only 1 get plasmid, the other one get nothing.
What could be the problem?
If your cells are not lysing then chances are that you didn't grow e.coli. I would check your cells before you redo the transformation to make sure they are not contaminated, also check the LB that you use to do your tranformation as well. Try doing a miniprep before growing the larger prep to save yourself the time of growing things that aren't e.coli (you may not get much DNA, but if your cells are lysing properly then at least you have a chance). good luck.
Howdy,
First, stupid question and I am sure it is standard in your lab protocols, but what is the concentration of Amp in your LB broth? Secondly, are these plasmids ones that have already been sequenced and used in your lab, or are they newly designed?
Typically, even if they are commonly used and already sequenced, you want to do a mini prep first on a number of colonies, confirm your region of interest by sequencing is in there how you like it, make freezer stock of that individual colony, and then use that stock to inoculate the Amp/LB for your maxi preps.....
This way you are not wasting $50 dollars for each failed maxiprep, and then come to find out the plasmid isn't functional or the plasmid of interest.