Transformation Dilemma - I hope if you can figure out (Nov/05/2008 )
I did transformation and obtained growth of E.Coli on the LB agar plates containing tetracyline and ampicillin. The E.Coli contain tetracyline and ampicilline resistant plasmids.
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
-SYDNEY-
QUOTE (SYDNEY @ Nov 6 2008, 12:33 AM)
I did transformation and obtained growth of E.Coli on the LB agar plates containing tetracyline and ampicillin. The E.Coli contain tetracyline and ampicilline resistant plasmids.
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
First, how old are your plates? They may not be selective any more. Antibiotics degrade quickly; I don't use anything older than 4 months. Also, if you have older plates, you can add Amp or Tet diluted in some LB to the top to make them more selective.
Second, did you have a negative control? (Plate cells which you take through the transformation protocol without adding vector.) Did you have more colonies on your experimental vs. the negative control plates?
Lastly, are you sure your plasmids are good? What is the source of the DNA?
Good luck!
-d.g.alina-
QUOTE (d.g.alina @ Nov 7 2008, 02:41 AM)
QUOTE (SYDNEY @ Nov 6 2008, 12:33 AM)
I did transformation and obtained growth of E.Coli on the LB agar plates containing tetracyline and ampicillin. The E.Coli contain tetracyline and ampicilline resistant plasmids.
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
First, how old are your plates? They may not be selective any more. Antibiotics degrade quickly; I don't use anything older than 4 months. Also, if you have older plates, you can add Amp or Tet diluted in some LB to the top to make them more selective.
Second, did you have a negative control? (Plate cells which you take through the transformation protocol without adding vector.) Did you have more colonies on your experimental vs. the negative control plates?
Lastly, are you sure your plasmids are good? What is the source of the DNA?
Good luck!
4 months? Wow. I rarely use Amp plate more than a month or so old!
Amp plates in particular are sensitive to light, so you need to keep them wrapped up at 4C.
I agree with dg: cells won't grow in media with antibiotics. Even satellite colonies on amp plates grow only after other cells deplete the local concentration of amp. Try the expt again with fresh plates.
-swanny-
QUOTE (d.g.alina @ Nov 6 2008, 10:41 AM)
QUOTE (SYDNEY @ Nov 6 2008, 12:33 AM)
I did transformation and obtained growth of E.Coli on the LB agar plates containing tetracyline and ampicillin. The E.Coli contain tetracyline and ampicilline resistant plasmids.
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
But when i released , there were no plasmids. Is it possible that teh competent cells of E.Coli dont take the plasmids and still they grow on the plates?
First, how old are your plates? They may not be selective any more. Antibiotics degrade quickly; I don't use anything older than 4 months. Also, if you have older plates, you can add Amp or Tet diluted in some LB to the top to make them more selective.
Second, did you have a negative control? (Plate cells which you take through the transformation protocol without adding vector.) Did you have more colonies on your experimental vs. the negative control plates?
Lastly, are you sure your plasmids are good? What is the source of the DNA?
Good luck!
Thank you for your reply.
We used one week plates, but i dont think the plates are a problem coz me and my firend did transformation with the same plates but she was doing her mutant and i was my wild type.
Second negative control? we really dont have a negative control in our lab coz these plasmids are gifted by some body to my supervisor. I am very new to this lab and it sbeen just 2 months i have been enrolled in PhD. nOBODY USE THE NEGATIVE CONTROL HERE.
The plasmids were of human GABAA receptors. I think teh antibiotics and verything was ok since my friends got very good results with her mutant type.
May be its just about the plasmids that they are not good becuase we i when i purified these palsmids initially, the band on gel were very faint. so what do you think
plasmids with what band intensity should be used for transformation>
I will be waiting for you reply.
-SYDNEY-
QUOTE (SYDNEY @ Nov 7 2008, 12:33 AM)
Thank you for your reply.
We used one week plates, but i dont think the plates are a problem coz me and my firend did transformation with the same plates but she was doing her mutant and i was my wild type.
Second negative control? we really dont have a negative control in our lab coz these plasmids are gifted by some body to my supervisor. I am very new to this lab and it sbeen just 2 months i have been enrolled in PhD. nOBODY USE THE NEGATIVE CONTROL HERE.
The plasmids were of human GABAA receptors. I think teh antibiotics and verything was ok since my friends got very good results with her mutant type.
May be its just about the plasmids that they are not good becuase we i when i purified these palsmids initially, the band on gel were very faint. so what do you think
plasmids with what band intensity should be used for transformation>
I will be waiting for you reply.
We used one week plates, but i dont think the plates are a problem coz me and my firend did transformation with the same plates but she was doing her mutant and i was my wild type.
Second negative control? we really dont have a negative control in our lab coz these plasmids are gifted by some body to my supervisor. I am very new to this lab and it sbeen just 2 months i have been enrolled in PhD. nOBODY USE THE NEGATIVE CONTROL HERE.
The plasmids were of human GABAA receptors. I think teh antibiotics and verything was ok since my friends got very good results with her mutant type.
May be its just about the plasmids that they are not good becuase we i when i purified these palsmids initially, the band on gel were very faint. so what do you think
plasmids with what band intensity should be used for transformation>
I will be waiting for you reply.
You don't need a large amount of vector for successful transformation. 100pg - 1ug for 50ul of compotent cells is the recommended range; I recommend adding in the nanogram range. You show make sure the identity of the DNA (plasmids) you isolated (did you miniprep?). I suggest digesting you isolated DNA with a restriction enzyme that is known to cut them and running this on a gel. The band should be the right size, etc.
-d.g.alina-
QUOTE (d.g.alina @ Nov 7 2008, 09:46 AM)
QUOTE (SYDNEY @ Nov 7 2008, 12:33 AM)
Thank you for your reply.
We used one week plates, but i dont think the plates are a problem coz me and my firend did transformation with the same plates but she was doing her mutant and i was my wild type.
Second negative control? we really dont have a negative control in our lab coz these plasmids are gifted by some body to my supervisor. I am very new to this lab and it sbeen just 2 months i have been enrolled in PhD. nOBODY USE THE NEGATIVE CONTROL HERE.
The plasmids were of human GABAA receptors. I think teh antibiotics and verything was ok since my friends got very good results with her mutant type.
May be its just about the plasmids that they are not good becuase we i when i purified these palsmids initially, the band on gel were very faint. so what do you think
plasmids with what band intensity should be used for transformation>
I will be waiting for you reply.
We used one week plates, but i dont think the plates are a problem coz me and my firend did transformation with the same plates but she was doing her mutant and i was my wild type.
Second negative control? we really dont have a negative control in our lab coz these plasmids are gifted by some body to my supervisor. I am very new to this lab and it sbeen just 2 months i have been enrolled in PhD. nOBODY USE THE NEGATIVE CONTROL HERE.
The plasmids were of human GABAA receptors. I think teh antibiotics and verything was ok since my friends got very good results with her mutant type.
May be its just about the plasmids that they are not good becuase we i when i purified these palsmids initially, the band on gel were very faint. so what do you think
plasmids with what band intensity should be used for transformation>
I will be waiting for you reply.
You don't need a large amount of vector for successful transformation. 100pg - 1ug for 50ul of compotent cells is the recommended range; I recommend adding in the nanogram range. You show make sure the identity of the DNA (plasmids) you isolated (did you miniprep?). I suggest digesting you isolated DNA with a restriction enzyme that is known to cut them and running this on a gel. The band should be the right size, etc.
Yeah i do miniprep.
i digested with teh restriction enzyme, i found some faint bands in the right size but may be dye to small quantity of plasmids, it got lost during the purification stage.
I was thinking to use some good plasmids and transform again and see what happens.
-SYDNEY-