mini-prep plasmid isolation by alkaline lysis
Posted 29 December 2010 - 07:17 AM
solution I: Tris-HCl, EDTA, RNAseH- resuspending the bacterial pellet after cfg 12000rpm/1'at RT
solution II: NaOH, SDS- inverting tubes several times, 3-5' incubation at RT
solution III: potassium acetate- inverting tubes several times,10'incubation on ice, and cfg at 12000rpm/10'at RT
after this I have to transfer supernatant to new tubes with ice cold isopropanol, cfg again and then resuspend the pellet with ice-cold 70% EtOH and cfg again, throw the supernatant and leave to dry, add deH20 or TE-buffer.
The thing that is bothering me is the part when I have to transfer supernatant to new tubes with isopropanol:
after cfg I got a white pellet containing the cell debris, rest of proteins and chromosomal DNA, but when I try to transfer the supernatant some sticky, spider net like colorless strings making it hard for me-is this the genomic or plasmid DNA?
Also, I am afraid not to collect this whithish pellet, so can I take it out with the sterile toothpick or tips, without to be afraid I will leave to much genomic DNA?
Thank you in advance
Posted 29 December 2010 - 07:33 AM
Posted 29 December 2010 - 11:36 PM
also, I would like to know for how long can I store the solutions (maximum:6months, or)? I am keeping them in +4degrees room. I know that SDS tends to precipitate so I pre-warm it in water-bath on around +40degrees, but what is with other reagents, do they have some "special needs"?
Edited by lab_microbe, 29 December 2010 - 11:51 PM.
Posted 30 December 2010 - 05:02 AM
Posted 30 December 2010 - 12:22 PM
Posted 31 December 2010 - 12:16 AM