- December 4, 2013 at 12:32 pm #17670nedalParticipant
i have two questions, which make s me confuse, would please help me to understand them:
You have a plasmid that is 5500 bp in size.
What is it’s molecular weight?
If you have a prep that is 1 mg/ml, what volume would you add to get 1 picomole?
100 microliters of buffer containing 0.25 micrograms of plasmid DNA was added to 400 microliters of competent cells. The mixture was heat shocked and added to 4.5 milliliters of L Broth. Subsequently 50 microliters of the L Broth was plated onto medium which selects for cells carrying the plasmid and the following day 256 colonies were counted on the plate. What was the transformation frequency?
i will be thankful
- December 4, 2013 at 8:12 pm #114825JackBeanParticipant
1 bp weights on average 650 Da (e.g. here: https://www.neb.com/tools-and-resources … -acid-data ). Thus 5.5 kbp plasmid weights 5500*650 Da.
If you know the MW of the plasmid, you should know how much 1 pmol weights (in mass units) ad thus it should be easy to calculate the volume when you know the concentration.
That’s just a lots of dilutions. In the beginning you have 0.25 ug/100ul = 250 ng/100ul = 2.5 ng/ul. Which is quite useless for you 😀
OK, so you have 0.25 ug and you dilute it as follows: 100 ul to fin. volume 500 ul (i.e. 5×). That added to fin. volume 5 ml (i.e. 10×). From that you take 50 ul, which is 1/100th of 5 ml, i.e. 2.5 ng of DNA. and this gave 256 colonies. thus you have efficiency about 100 colonies per 1 ng of DNA if I calculate correctly.
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