6 replies to this topic

[lab_microbe]

I am doing the alkaline lysis procedure using O/N cultures of E.coli to extract the plasmid and using:

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

[phage434]

The stringy stuff is genomic DNA.  If you having trouble with taking the supernatent, likely you are using too many cells as input to the prep.  Either increase the volume of your resuspension and lysis buffers, or reduce the pellet size.  You want just the clear liquid without the stringy material.

[lab_microbe]

phage434, on 29 December 2010 - 07:33 AM, said:

The stringy stuff is genomic DNA.  If you having trouble with taking the supernatent, likely you are using too many cells as input to the prep.  Either increase the volume of your resuspension and lysis buffers, or reduce the pellet size.  You want just the clear liquid without the stringy material.

thank you

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.

[phage434]

The RNAseH in buffer 1 is perhaps sensitive to temperature, although when you try to get rid of it, it doesn't behave that way.  The NaOH in buffer 2 will eventually absorb CO2 from the air and form sodium bicarbonate.  If you keep things closed and at +4, I would say things are good for a year or more.  Make sure your 70% ethanol solution is tightly closed.  Evaporation will change the percentage, which can produce problems in the pellet wash step.

[HomeBrew]

You also want to be very gentle at the lysis step -- the idea is to gently lyse the cells so the small plasmids leak out while the big chromosome remains trapped and is thus removed with the cell debris.  Invert the tubes a few times at this step and move on -- no vortexing.

[kajmak]

After solution 3 add the same volume (as for solution 3) of chloroform. Skip all incubations, just invert tubes after solution 2 and chloroform. Centrifuge for 4-5 min at 14 000 rpm and you should be able to get nice and clean supernatant.

[lab_microbe]

 thank you all for your kind advices  

bests