dilute the ligation reaction for electroporation - (Jun/18/2008 )
I do so many time the electroporation for BL21 E.coli by 1ul direct ligation but in electroporator always flash come so is it mportant to dilute the ligation reaction and what the best way to dilute it.............
-longer-
QUOTE (longer @ Jun 18 2008, 01:06 PM)
I do so many time the electroporation for BL21 E.coli by 1ul direct ligation but in electroporator always flash come so is it mportant to dilute the ligation reaction and what the best way to dilute it.............
Yep, dilute it with ddH20 10 times, and use 1ul.
Electroporation is very efficient method (Vs. heat shock), so using diluted ligation reaction will not be a problem. In fact, in my experience, it incereses the efficiency.
-cellcounter-
QUOTE (cellcounter @ Jun 18 2008, 11:20 PM)
QUOTE (longer @ Jun 18 2008, 01:06 PM)
I do so many time the electroporation for BL21 E.coli by 1ul direct ligation but in electroporator always flash come so is it mportant to dilute the ligation reaction and what the best way to dilute it.............
Yep, dilute it with ddH20 10 times, and use 1ul.
Electroporation is very efficient method (Vs. heat shock), so using diluted ligation reaction will not be a problem. In fact, in my experience, it incereses the efficiency.
thank u for ur idea today i did transformation with diluted mixture and no flash
and tommorow I will check the no. of colony thank u 
-longer-
QUOTE (longer @ Jun 19 2008, 04:42 PM)
QUOTE (cellcounter @ Jun 18 2008, 11:20 PM)
QUOTE (longer @ Jun 18 2008, 01:06 PM)
I do so many time the electroporation for BL21 E.coli by 1ul direct ligation but in electroporator always flash come so is it mportant to dilute the ligation reaction and what the best way to dilute it.............
Yep, dilute it with ddH20 10 times, and use 1ul.
Electroporation is very efficient method (Vs. heat shock), so using diluted ligation reaction will not be a problem. In fact, in my experience, it incereses the efficiency.
thank u for ur idea today i did transformation with diluted mixture and no flash
and tommorow I will check the no. of colony thank u daer cellcounter
am not happay because even in control plate no colony what can I do
-longer-
A suggestion...Float small milipore filter paper on dd-water in a petri-dish..and simply put 5-10uL of your ligation mixture on them for an hour or so...After that recover the ligation mix with a pipette...and go for transformation...Principle as you might have guessed by now is "micro" dialysis for desalting..hopefully it works...do let me know..
-cool_hotlad-
QUOTE (cool_hotlad @ Jun 20 2008, 05:34 AM)
A suggestion...Float small milipore filter paper on dd-water in a petri-dish..and simply put 5-10uL of your ligation mixture on them for an hour or so...After that recover the ligation mix with a pipette...and go for transformation...Principle as you might have guessed by now is "micro" dialysis for desalting..hopefully it works...do let me know..
This is a good idea.
1. There are several other things that may be going wrong, so, you need to troubleshoot other aspects too. Starting from the bacterial strain, media, antibiotics, electroporator, cuvettes, incubation times etc etc. As you are already doing control plasmid electroporation, you may be fine without troubleshooting cloning part (ligation etc), but make sure the controls you are doing cover every eventuality.
More on bacterial electroporation: http://search.vadlo.com/b/q?p=1&sn=158...rel=0&srt=0
..
-cellcounter-
It is a very bad idea to try to transform BL21 strains directly with a ligation product. You will save time and grief by transforming first into DH5a, DH10B or another cloning strain, doing a miniprep, and transforming the BL21 with the plasmid DNA. BL21 has very poor transformation efficiency.
-phage434-
QUOTE (phage434 @ Jun 21 2008, 12:02 AM)
It is a very bad idea to try to transform BL21 strains directly with a ligation product. You will save time and grief by transforming first into DH5a, DH10B or another cloning strain, doing a miniprep, and transforming the BL21 with the plasmid DNA. BL21 has very poor transformation efficiency.
Hi,
thanks when I try with top10f I got result with one plasmid
but still I have problem with the second plasmid accutally its Euc. vectors did u have expri. in the EU vectors.-longer-