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site-directed mutagenesis


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20 replies to this topic

#16 Trof

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Posted 18 March 2013 - 07:24 AM

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.

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#17 zzha158

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Posted 18 March 2013 - 03:55 PM

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.

#18 zzha158

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Posted 18 March 2013 - 04:01 PM

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.

#19 GNANA

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Posted 19 March 2013 - 01:26 AM

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...
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#20 zzha158

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Posted 19 March 2013 - 10:25 PM

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

#21 GNANA

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Posted 20 March 2013 - 12:55 AM

Your cell issue doesnt exclude the PCR as working, so as trof suggested do a pcr in parallel to test the efficiency of PCR and proceed in new cells. good luck...
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