transformation - urgent (Jun/15/2006 )
I am doing priliminary practical on transforming in which I am trying to transform plasmid pUC18 to E coli HB101. I need a useful proctocol. please help me sending it.
The amount of DNA to be used depends on the transformation efficiency of the cells.
You can do as follows (always worked in my hands):
- Thaw the cells
- Put 100ul of cells (even here it depends, you could use 30ul) in a sterile eppendorf
- Add your DNA
- Incubate 30 min on ice
-Incubate 2 min at 37 degrees
- Incubate 10 min on ice
- Plate the bacteria
do you have competent bacteria?
If no, then you have to make competent bacteria.
inoculate 500 uL of bacteria in 50 mL LB medium
let grow 3-4 hours until OD600 reaches 0.3
take 30 mL of these bacteria, put in a 50 mL Falcon tube.
Let sit at 4°C for 15 min
centrifuge 5000 rcf, 4°C, 10 min
put the pellet on ice (it's very important to do everything at 4°C)
prepare fresh Tris HCl pH8.0 10 mM; CaCl2 50 mM. chill the solution on ice.
resuspend the pellet in 15 mL of tris-CaCl2, mix in ice. It must stay cold.
5 minutes on ice.
centrifuge 10 minutes at 5000 rcf, at 4°C
discard the supernatant
resuspend the pellet in 1 mL of tris-CaCl2
your bacteria are competent. you can now transform them.
add 50 ng of plasmid to 200 uL of bacteria. let sit 20 minutes on ice.
incubate 90s at 42°C
add 800 uL of LB. incubate 30 minutes à 37°C.
Inoculate an agar plate (sorry, not good english), let grow overnight
if you transform with a plasmid, I would only inoculate one tenth of your bacteria, because it's very efficient.( it means inoculate only 100 uL out of 1 mL of bacteria)
if you transform a ligation product, it's less efficient. I would centrifuge the bacteria (1 mL) and resuspend the pellet in 100 uL and inoculate 100 uL (i.e. all your bacteria).
all the best