Why not a SINGLE RECOMBINANT ? - (Jun/11/2006 )
I think the transformation and ligation are so weird!
Before I did the transformation, everything is ok, when I did the electrophoresis, both the interested dna and vector is very bright
And I also use the simple sticky ligation (EcoRI/MluI), but after I did the transformation and ligation, none of the recombinant was grown! It has happened so many times!
I checked:
1. It is the Amp, not kan
2. The complete cell is ok
3. The T4 liganase is ok
4. The plate is also ok
5. The time and temp of the incubation box is also ok.
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~ 
-sunnylyon-
QUOTE (sunnylyon @ Jun 11 2006, 07:28 AM)
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
well you have to find it yourself, in Molecular biology strange things always hapens, in my lab 6 base cutters dont work we did every thing change anything that was possible buy new Items no answer until we tried in another lab and no one can really explain why!
-akhshik-
QUOTE (sunnylyon @ Jun 11 2006, 06:28 AM)
I think the transformation and ligation are so weird!
Before I did the transformation, everything is ok, when I did the electrophoresis, both the interested dna and vector is very bright
And I also use the simple sticky ligation (EcoRI/MluI), but after I did the transformation and ligation, none of the recombinant was grown! It has happened so many times!
I checked:
1. It is the Amp, not kan
2. The complete cell is ok
3. The T4 liganase is ok
4. The plate is also ok
5. The time and temp of the incubation box is also ok.
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
Before I did the transformation, everything is ok, when I did the electrophoresis, both the interested dna and vector is very bright
And I also use the simple sticky ligation (EcoRI/MluI), but after I did the transformation and ligation, none of the recombinant was grown! It has happened so many times!
I checked:
1. It is the Amp, not kan
2. The complete cell is ok
3. The T4 liganase is ok
4. The plate is also ok
5. The time and temp of the incubation box is also ok.
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
Did you check transformation was working by using a control vector?
-JPStewart-
what molar end ratios of vector: insert did you used? try changing those.
i generally use 1:3, 1:5 and sometimes 1:8
low or excess amount of insert may affect your ligation reaction
i hope it will help 
-dodosko-
QUOTE (JPStewart @ Jun 12 2006, 01:34 AM)
QUOTE (sunnylyon @ Jun 11 2006, 06:28 AM)
I think the transformation and ligation are so weird!
Before I did the transformation, everything is ok, when I did the electrophoresis, both the interested dna and vector is very bright
And I also use the simple sticky ligation (EcoRI/MluI), but after I did the transformation and ligation, none of the recombinant was grown! It has happened so many times!
I checked:
1. It is the Amp, not kan
2. The complete cell is ok
3. The T4 liganase is ok
4. The plate is also ok
5. The time and temp of the incubation box is also ok.
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
Did you check transformation was working by using a control vector?
Not really, I think. So what is the control vector composed of? Do u mean the vector which does not have the intrested DNA?
-sunnylyon-
QUOTE (dodosko @ Jun 12 2006, 08:30 AM)
what molar end ratios of vector: insert did you used? try changing those.
i generally use 1:3, 1:5 and sometimes 1:8
low or excess amount of insert may affect your ligation reaction
i hope it will help
i generally use 1:3, 1:5 and sometimes 1:8
low or excess amount of insert may affect your ligation reaction
i hope it will help
Thank you very much! well, I used 1:3 for the sticky-end ligation, and 1:5or 1:10 for blunt-end ligation, and in this case ,it is a sticky-end ligation, so I used 1:3, but still can not work, sigh....
-sunnylyon-
QUOTE (dodosko @ Jun 12 2006, 08:30 AM)
what molar end ratios of vector: insert did you used? try changing those.
i generally use 1:3, 1:5 and sometimes 1:8
low or excess amount of insert may affect your ligation reaction
i hope it will help
i generally use 1:3, 1:5 and sometimes 1:8
low or excess amount of insert may affect your ligation reaction
i hope it will help
Thank you very much! well, I used 1:3 for the sticky-end ligation, and 1:5or 1:10 for blunt-end ligation, and in this case ,it is a sticky-end ligation, so I used 1:3, but still can not work, sigh....
-sunnylyon-
QUOTE (akhshik @ Jun 11 2006, 10:54 PM)
QUOTE (sunnylyon @ Jun 11 2006, 07:28 AM)
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
well you have to find it yourself, in Molecular biology strange things always hapens, in my lab 6 base cutters dont work we did every thing change anything that was possible buy new Items no answer until we tried in another lab and no one can really explain why!
Do u mean that, when u change to another lab, u can cut the 6 base:)?
-sunnylyon-
QUOTE (JPStewart @ Jun 12 2006, 12:50 PM)
QUOTE (sunnylyon @ Jun 11 2006, 06:48 PM)
QUOTE (JPStewart @ Jun 12 2006, 01:34 AM)
QUOTE (sunnylyon @ Jun 11 2006, 06:28 AM)
I think the transformation and ligation are so weird!
Before I did the transformation, everything is ok, when I did the electrophoresis, both the interested dna and vector is very bright
And I also use the simple sticky ligation (EcoRI/MluI), but after I did the transformation and ligation, none of the recombinant was grown! It has happened so many times!
I checked:
1. It is the Amp, not kan
2. The complete cell is ok
3. The T4 liganase is ok
4. The plate is also ok
5. The time and temp of the incubation box is also ok.
Then why can not grow a single recombinant? I cannot guess why, but I know there is one reason.
As one said, if the experiment can not prove what you want, it must prove sth else, but what is the STH?
Would u please tell me? So thanks!~
Did you check transformation was working by using a control vector?
Not really, I think. So what is the control vector composed of? Do u mean the vector which does not have the intrested DNA?
-JPStewart-
How abt the competent cell's efficiency? Try transforming uncut vector, just to confirm efficiency.
-scolix-