2 replies to this topic

[sc7]

Hi
im having trouble calculating the transformation efficiency for the competent cells. this is what i was given:

5 µl pUC18 (0.1 ng/ µl)
50 µl E. coli competent cells
Left on ice 30 minutes
Heat shocked at 42C for 45 seconds
Added 500 µl NZY broth
Place in 15 ml culture tube in shaking incubator 37C for 1 hr
Then spread 10 µl, 50 µl and 100 µl on different LB amp plates.

10 µl gave 495 colonies.

would really appreciate it if someone could help me out, the answer should be in cfu/ug units. im mainly uncertain on the amount of DNA that was in the plate.

thanks!
-sc7-

[San Diego Mike]

If you have 5 ul @ 0.1 ng/ ul, then you are starting with 0.5 ng of pUC18.
Once you add 500 ul NZY broth, your total volume is 5 ul + 50 ul + 500 ul = 555 ul.
And the 555 ul contains 0.5 ng of pUC18, so the concentration is 0.5 ng/555 ul = 0.0009 ng/ul.
If you spread 10 ul of 0.0009 ng/ul pUC18-containing solution on the plate, you are spreading 0.009 ng of pUC18. Converting to ug, this is 0.000009 ug.

If this 10 ul gave 495 colonies, you get 495 cfu / 0.000009 ug = XXXXX

(I left off the answer because I want to be sure you understand the reasoning - not just copy the answer - although I am sure that you would not do that   ).

Hope this helps.

Mike
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im having trouble calculating the transformation efficiency for the competent cells. this is what i was given:

5 µl pUC18 (0.1 ng/ µl)
50 µl E. coli competent cells
Left on ice 30 minutes
Heat shocked at 42C for 45 seconds
Added 500 µl NZY broth
Place in 15 ml culture tube in shaking incubator 37C for 1 hr
Then spread 10 µl, 50 µl and 100 µl on different LB amp plates.

10 µl gave 495 colonies.

would really appreciate it if someone could help me out, the answer should be in cfu/ug units. im mainly uncertain on the amount of DNA that was in the plate.

thanks!
-sc7-
[/quote]

[sc7]

thanks alot Mike! i now understand what you did