How to elude DNA sample from a paper? - and then transform E.coli... (Feb/25/2006 )
I have just registered to this forum and I am really in trouble . I am trying to elude a plasmid sample from an ordinary paper (not filter paper). The sender told me that they loaded 100ng of sample (mixed with a small amount of loading dye) on the paper and let it dry. I cut the paper of about 5mm diameter, kept it in 50 uL TE buffer for about 3 hours and then used 10 uL of it to transform 100uL of competent E.coli cells. I have already tried three E.coli transformations and lost great amount of sample. The competent E.coli that I used is working for ordinary plasmid transformations, for example, I got 14 colonies after transformation with 1uL of another plasmid with a concentration of 120ng/uL. I had prepared the competent E.coli cells by using ordinary CaCl2 treatments. What can be the problem do you think? Can it be that the cells are not competent enough to be transformed with very low concentrations of plasmid. What can I do to elude plasmid more effectively from paper and would it be better to use electrocompetent cells and transform them via electroporation?
Thanks for your helps in advance..
So at your efficiency you only see 14 colonies with 120ng of plasmid... this means that with your 100ng spot of plasmid.. even assuming 100% came off the paper you have 100ng/50ul or 2ng/ul so transformed 20ng so with your efficiency, you can only expect to see at most 2.35 colonies?? This completely disregards contamination with loading buffer etc and the effect of that on transformation.. not to mention that it is unlikely that you got 100% of the plasmid back out of the paper...
So to sum up I think you need better competent cells.. your efficiency with pure plasmid is not high enuf.. It will be hard to get colonies... usually commercial competent cells have a cfu of 10^8/ug (100,000,000 colonies per ug or 100,000 per ng) or so and you would easily see colonies at your concentrations with these cells.
So I should find a way to prepare better competent cells than, thanks beccaf22..