site-directed mutagenesis - (Feb/28/2013 )
GNANA on Mon Mar 11 07:34:38 2013 said:
Are you sure the PCR worked ? what about the efficiency of the competent cells you using ?
I dont use the quick change lit. How do I test it ?
you dnt necessarily need to use the kit, can very well do site directed mutagenesis using a High fidelity pol/mix and the Dpn, + good competent cells obviously...is your question is about, how to test the efficiency of competent cells? if so you will find enough info in this link..http://www.protocol-online.org/forums/topic/28507-transformation-problem/page__hl__transformation
GNANA on Thu Mar 14 17:05:07 2013 said:
you dnt necessarily need to use the kit, can very well do site directed mutagenesis using a High fidelity pol/mix and the Dpn, + good competent cells obviously...is your question is about, how to test the efficiency of competent cells? if so you will find enough info in this link..http://www.protocol-..._transformation
Thank you for your help. I did a positive control last week. It seemed that the amount of DNA was low (our home-made cells are not that competent), I increased the DNA up to ~400 ng, and the transformation succeeded. Now I am just waiting for the sequencing report. If the sequencing reports no mutation as I wanted, what should I do next. Keep on trying or change the protocol.
I really appreciate your help. At the moment, my supervisor is admitted in hospital and this is my first time to use this technique.
Sequencing shows no mutation at that site, it is really frustrating. I have been doing this for months. Is it due to primer?
i dnt think it is because of ur primers, getting transformants in 400ng says ur cells are not efficient enough....i would suggest you to redo the cells and proceed only when you get good number of transfomants at 10pg conc of plasmid.
I think it may mean your primers don't work. Because by DpnI you cut the wild type plasmid, if your reaction was running, only the mutant in excess would remain and transform. But if you only get transformants in 400ng, where there are traces of wild-type plasmid in such amount, cells are transformed with wild-type only.
It's not usual to see plasmid on gel with only 18 cycles or so, so to test efficiency of PCR increase the number of cycles to be able to see some product. If you don't see any you may try to optimize the PCR.
GNANA on Mon Mar 18 09:54:34 2013 said:
i dnt think it is because of ur primers, getting transformants in 400ng says ur cells are not efficient enough....i would suggest you to redo the cells and proceed only when you get good number of transfomants at 10pg conc of plasmid.
10pg? thats tiny amount. I will ask the technician to prepare some cells. For the positive control, I cant even get one colony in 200 ng.
Trof on Mon Mar 18 15:24:16 2013 said:
I think it may mean your primers don't work. Because by DpnI you cut the wild type plasmid, if your reaction was running, only the mutant in excess would remain and transform. But if you only get transformants in 400ng, where there are traces of wild-type plasmid in such amount, cells are transformed with wild-type only.
It's not usual to see plasmid on gel with only 18 cycles or so, so to test efficiency of PCR increase the number of cycles to be able to see some product. If you don't see any you may try to optimize the PCR.
Yeah I used 18 cycles following the protocol from mutagenesis quick change kit, After PCR, I treated PCR product (concentration measured by photometer) with DPNI (4U) in NEB buffer 4 for 90 min at 37.
Thats what i am concerned abt ur cells, i would definitely wont use those cells even for normal transformation that gives no colony in 200ng input....and yes you shd test the efficiency in such lower amount of plasmid. In my experience i successfully got cloning-transformants and PCR-transformants in the cells that gave me around 50 colonies in 100pg input in the efficiency test. so in my hands, i consider this as the minimal efficiency required for these procedures. so i think ur cells are way down the required competency. check it out...
GNANA on Tue Mar 19 09:26:49 2013 said:
Thats what i am concerned abt ur cells, i would definitely wont use those cells even for normal transformation that gives no colony in 200ng input....and yes you shd test the efficiency in such lower amount of plasmid. In my experience i successfully got cloning-transformants and PCR-transformants in the cells that gave me around 50 colonies in 100pg input in the efficiency test. so in my hands, i consider this as the minimal efficiency required for these procedures. so i think ur cells are way down the required competency. check it out...
OK. Thank you for your suggestion. Other PIs offered me some XL-blue super competent cells but a bit old . I will try those first