2 replies to this topic

[bacchess]

Hi! I have a few questions:

1. Does too high a concentration of DNA hinder heat pulse transformation of XL1 Blue? If so, what is the optimum concentration?

2. I have a set of primers which doesn't seem to work. Do you know any website to check the feasibility of these primers? I know dimerization of primers, formation of hairpins and Tm affects the primer's efficiency but I don't know the exact guidelines for these criteria (acceptable # of self-dimerizing bp, optimum Tm, etc.).

Thanks a bunch!

[pcrman]

Quote

Does too high a concentration of DNA hinder heat pulse transformation of XL1 Blue? If so, what is the optimum concentration?

Yes, the quantity of DNA used in transformation will influence the transformation efficiency. The transformation efficiency usually decreases with an excess of DNA. For example, when transforming 10pg plasmid DNA, the efficiency of Top10 cells is 1.6x10^8 while transforming 1ng the efficiency is 1.4 10^7 and transforming 1ug the efficiency is 7.2x 10^4.

So use no more than 1 ug DNA for a transformation reaction.

Quote

Do you know any website to check the feasibility of these primers?

yes, many online primer design tools allow you to do this. such as:
Primer3 http://frodo.wi.mit....primer3_www.cgi

or check this page:
http://www.bioinformatics.vg/biolinks/bioi...520Design.shtml

[bioforum]

These threads may be of some help.

http://www.protocol-online.org/forums/inde...p?showtopic=699

http://www.protocol-online.org/forums/inde...?showtopic=2969