DNA extraction from E. coli - solutions needed? (Mar/20/2011 )

I am currently looking into the extraction of DNA from E. coli.

I want to extract the DNA using a manual method and also using centrifuge.

Once I have extracted the DNA I want to test it for yield, purity and possibly run gel electrophoresis to determine molecular weight.

I have found some protocols online but I am stuck on a few things. Uses the centrifuge method a couple of this are not well explained.

They use a Lysis solution which is SDS and also use RNase solution. But the protocol also uses a Protein Precipitation Solution (PPS) and a DNA Rehydration Solution (RH) but does not say what it is made up of.

Can anyone help clear this up for me?

As once the DNA has been extracted I want to test for purity in the spectrophotometer.

Thanks
Rezarf

Actually, if you just want to PCR to clone a gene, a tip-touch method is more than sufficient: use a tip, touch a colony and suspend into PCR tube with primers and master mix.

Protein precipitate solutions usually is ammonium acetate and DNA Rehydration solution usually is TE buffer.

adrian kohsf on Sun Mar 20 15:40:55 2011 said:

Welcome, and all the best in your work.

Also for protein precipitation you can use sodium chloride at very high concentration ( for prepare just added NACl until no more in solution). When doing the extraction at the step of PP incubate on ice for 5 min before centrifuge.

The lab tech says they do not have Ammonium Acetate nor does it have phenol:chloroform so looks like it will be sodium chloride. Should I add a protease to break down the proteins?

So once i add the PP do i then incubate on ice for 5 mins before centrifuge?

Thanks
Rezarf

I not sure about Sodium chloride. Do you have Sodium acetate or any "*acetate"? perhaps it might be able to substitute...
Can you refer me to the link?
Yes, "usually" you have to incubate on ice before centrifuge for that step, if I remember correctly.