Isolating intact chromosomal DNA from E. coli - (Mar/29/2007 )
Hi I am trying to isolate chromosomal DNA of E. coli to run sucrose gradients. Phenol extraction seems to break the DNA, I also tried some comercial kits, but they also shear the DNA. Is there any way to extract intact chromosomal DNA from E. coli?
The standard approach is to embed the cells in agarose and then digest the cells in-place with a mixture of lauryl sarcosine and proteinase-K in EDTA at 50C. The embedded cells are lysed and the DNA released, but kept in place. The DNA can be used in this form for digestion or pulsed-field gels. If free DNA is needed, digestion of the agarose with beta-agarase is possible, but the DNA is almost impossible to maintain in an undamaged form when floating freely in liquid. Pipetting is impossible.
OH, and how about extracting the chromosomal DNA only for PCR ?
I am working in chem lab, and I need only one gene.........thus I don't want to buy 1 kit with 50 or 100 reaction where I just use only one reaction.........
I am wonder, if colony PCR work, could I just directly add cells to the PCR tube and run my reaction ?
If you just want to do PCR of a single gene, then adding cells directly to the PCR reaction is the way to go. Use a small pipet tip to take a very small amount of a single colony and resuspend the cells in 50 ul of water and vortex. Use 0.5 ul of this in a 20 ul PCR reaction. Extend the initial 95C denaturing phase of the PCR reaction to 5 minutes. Too many cells is the normal way for people to get this wrong. Cycle for 35-40 cycles.