Hi,
Wish you baskets of smile :D :)
Last time, I explained about, Electrocompetent Cells of DH5 alpha. As a continuation, this time you are gonna know about, transforming those electrocompetent cells with Plasmids. The transformation technique is very simple.
Last time I didn't explain you about the procedure to prepare electrocompetent cells. Let me give you a short
procedure.
Procedure to prepare electrocompetent cells:
1) Inoculate the E.coli DH5 alpha cells the previous day in LB broth.
2) Subculture it, the next day such that you have initial O.D (Optical Density) of 0.05
3) After two hours, you must have an O.D of 0.6 to 1
(This is because, the cells will be in log phase or in active metabolic state in this O.D)
4) Then, ultra centrifuge your culture to separate the cells.
5) Ultra Centrifuge twice with cold water and one time with glycerol.
6000 rpm for 7 minutes at 4 degree c with cold water
4000 rpm for 5 minutes using glycerol.
6) Now, your electrocompetent cells are ready, put them in eppendorfs, store them in deep freezer with liquid nitrogen for further use ( - 80 degree C)
( I was very much exited when I saw the liquid nitrogen and deep freezer for the first time :)
That's why I always enjoy my lab hours)
Note:
The exact mechanism of altering the cell permeability by this procedure is not known.
The centrifugation with cold water is done for removing salts and ions present in the cell.
Glycerol acts as a cryoprotectant, maintains osmotic pressure.
Okay, lets go for transformation procedure now.
Things you need for this transformation are:
Electroporation cuvettes, Electroporation system, Laminar air flow hood, incubator.
These are the little sophisticated things you need apart from, Pipettes, petriplates, culture tubes etc.,
Procedure:
1) Thaw your electrocompetent cells stored at -80 degree C using ice.
2) For 20 micro liters of Cell, use 100 to 200 nanogram of plasmid DNA, mix and tap, leave in ice, for15 minutes.
3) Transfer this content from eppendorfs to electroporation cuvettes, and give pulse using electroporation system. ( During this electric shock, the DNA will enter into your electrocompetent cells)
4) Wash your cuvettes with LB broth and then tranfer the content to culture tubes else you can also use the cuvette during the incubation.
5) Use the Electro competent cells as your Control. (Give shock to these cells also, though they don't have DNA to get transformed)
Incubate your cells for 1 hour under appropriate conditions before plating.
6) Plate your Cells and cells+DNA using LB ampicillin plates (Spread plate method)
Observe your plates the next day after incubation,
The cell + Plasmid DNA (with ampicillin resistant marker) plate will show growth, if transformation is complete.
The Cell alone plate won't have growth.
I asked you to incubate your cells before plating them. you know why??
Because your transformed cell won't be having enough plasmid to resist your ampicillin. So, if you give them an hour, their pDNA will replicate and produce enough copies to produce enough beta lactamase.
You need detailed procedure?
Then comment your mail i.d.
I'l send you.
Any doubts, queries? mistakes? feel free to comment.
Wish you baskets of smile :D :)
Last time, I explained about, Electrocompetent Cells of DH5 alpha. As a continuation, this time you are gonna know about, transforming those electrocompetent cells with Plasmids. The transformation technique is very simple.
Last time I didn't explain you about the procedure to prepare electrocompetent cells. Let me give you a short
procedure.
Procedure to prepare electrocompetent cells:
1) Inoculate the E.coli DH5 alpha cells the previous day in LB broth.
2) Subculture it, the next day such that you have initial O.D (Optical Density) of 0.05
3) After two hours, you must have an O.D of 0.6 to 1
(This is because, the cells will be in log phase or in active metabolic state in this O.D)
4) Then, ultra centrifuge your culture to separate the cells.
5) Ultra Centrifuge twice with cold water and one time with glycerol.
6000 rpm for 7 minutes at 4 degree c with cold water
4000 rpm for 5 minutes using glycerol.
6) Now, your electrocompetent cells are ready, put them in eppendorfs, store them in deep freezer with liquid nitrogen for further use ( - 80 degree C)
( I was very much exited when I saw the liquid nitrogen and deep freezer for the first time :)
That's why I always enjoy my lab hours)
Note:
The exact mechanism of altering the cell permeability by this procedure is not known.
The centrifugation with cold water is done for removing salts and ions present in the cell.
Glycerol acts as a cryoprotectant, maintains osmotic pressure.
Okay, lets go for transformation procedure now.
Things you need for this transformation are:
Electroporation cuvettes, Electroporation system, Laminar air flow hood, incubator.
These are the little sophisticated things you need apart from, Pipettes, petriplates, culture tubes etc.,
Procedure:
1) Thaw your electrocompetent cells stored at -80 degree C using ice.
2) For 20 micro liters of Cell, use 100 to 200 nanogram of plasmid DNA, mix and tap, leave in ice, for15 minutes.
3) Transfer this content from eppendorfs to electroporation cuvettes, and give pulse using electroporation system. ( During this electric shock, the DNA will enter into your electrocompetent cells)
4) Wash your cuvettes with LB broth and then tranfer the content to culture tubes else you can also use the cuvette during the incubation.
5) Use the Electro competent cells as your Control. (Give shock to these cells also, though they don't have DNA to get transformed)
Incubate your cells for 1 hour under appropriate conditions before plating.
Observe your plates the next day after incubation,
The cell + Plasmid DNA (with ampicillin resistant marker) plate will show growth, if transformation is complete.
The Cell alone plate won't have growth.
I asked you to incubate your cells before plating them. you know why??
Because your transformed cell won't be having enough plasmid to resist your ampicillin. So, if you give them an hour, their pDNA will replicate and produce enough copies to produce enough beta lactamase.
You need detailed procedure?
Then comment your mail i.d.
I'l send you.
Any doubts, queries? mistakes? feel free to comment.
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