If your control reaction transforms 10 ng of DNA, that is 10**-2 ug. If your cells had a transformation efficiency of 10**8 cfu/ug (which is typical for good chemically competent cells) you should get 10**-2 x 10**8 = 10**6 transformants. You are getting 200 instead, so this is your problem. Get cells that 5000 times more competent.
I used new cells and got thousands colonies colonies in the transformation control and got 83 transformant colonies with my insert. 83 is the higest number I have got so far.
you should limit the ligation to 50 ng or less of plasmid DNA, which given the molar ratios (you do know it is molar, not ng ratio!) of insert:vector will mean a lot less nsert than you are using.
I'm a bit confused about this. These molar ratios are of the final ligation mix?
I estimate the ng quantity of my purified PCR product using a 100 bp ladder, and by knowing that the vector is 50ng/ul I estimate the 3:1 ratio of respective ng in the ligation mix. How do I calculate the proper molarity?
Ok. Most of that should be fine, though you should limit the ligation to 50 ng or less of plasmid DNA, which given the molar ratios (you do know it is molar, not ng ratio!) of insert:vector will mean a lot less insert than you are using.
For pJET, the recommended PCR product quantity to obtain 0.15 pmol of DNA ends is 25-50 ng for products of 500-1000 (my product is 700 pb). Is not mentioned if is purified PCR product or not.
bob1, on 01 February 2013 - 02:53 PM, said:
With 10 ng of control transformation you should be getting several hundred colonies on the plate, there may be a problem with the transforamation efficiency of your bugs.
In my transformation control I get 200+ colonies (I use 1 ul of miniprep purified pGEM with an insert); quantity I don't get using my ligation product. Just few hundreds would be enough.