Team:Brown/Notebook Protocols/CFU

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Revision as of 00:31, 22 October 2009

How to calculate CFU’s

(pg plasmid DNA/total reaction volume) x plated volume = pg DNA plated

(avg # of colonies/pg DNA plated) x (10^6 pg / µg) = (# of transformed cells/ µg) (CFU)

Start by taking a tube of competent cells that you have prepared and add 100pg of plasmid DNA from any one of your miniprep samples.

Go through the transformation the same way that you would any other time.

I usually take the volume of transformed cells (100μL) and add the same volume of LB.

I then plate out a number of dilutions of the mix, 0.5 (100μL), 0.05 (10μL), and 0.005 (1μL) onto the appropriate selection media (you obviously can’t spread 10 or 1μL so add more LB on the plate to spread the cells effectively).

Let them grow up Overnight and count the plate that has between 20 and 200 colonies and go through the calculations.

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Compiled by Adrian Reich, July 2009

@@ Line 8: / Line 8: @@
-How to calculate CFU’s
+'''<big>How to calculate CFU’s</big>'''
 ----
-(pg plasmid DNA/total reaction volume) x plated volume = pg DNA plated
+'''(pg plasmid DNA/total reaction volume) x plated volume = pg DNA plated'''
-(avg # of colonies/pg DNA plated) x (10^6 pg / µg) = (# of transformed cells/ µg) (CFU)
+'''(avg # of colonies/pg DNA plated) x (10^6 pg / µg) = (# of transformed cells/ µg) (CFU)'''
-Start by taking a tube of competent cells that you have prepared and add 100pg of plasmid DNA from any one of your miniprep samples. Go through the transformation the same way that you would any other time. I usually take the volume of transformed cells (100μL) and add the same volume of LB. I then plate out a number of dilutions of the mix, 0.5 (100μL), 0.05 (10μL), and 0.005 (1μL) onto the appropriate selection media (you obviously can’t spread 10 or 1μL so add more LB on the plate to spread the cells effectively). Let them grow up O/N and count the plate that has between 20 and 200 colonies and go through the calculations.
+Start by taking a tube of competent cells that you have prepared and add 100pg of plasmid DNA from any one of your miniprep samples.
+Go through the transformation the same way that you would any other time.
+I usually take the volume of transformed cells (100μL) and add the same volume of LB.
+I then plate out a number of dilutions of the mix, 0.5 (100μL), 0.05 (10μL), and 0.005 (1μL) onto the appropriate selection media (you obviously can’t spread 10 or 1μL so add more LB on the plate to spread the cells effectively).
+Let them grow up Overnight and count the plate that has between 20 and 200 colonies and go through the calculations.
+---
+Compiled by Adrian Reich, July 2009