How to get a good quality of plasmid in maxi prep. - (Feb/23/2006 )

Hi,
I know plasmid maxi-prep. can be performed by Qiagen kit or CsCl2 gradient. Both of them can get a good quality of DNA. As the Kit seems expensive for us when 2 weeks or 1 week need a kit , and CsCl2 gradient purification is too complicated for me.

Do you know there are any other method can yield a good quality and quantity of plasmid DNA?
How about the PEG8000 purification? I just a little worry about the quality as I need to use these DNA for luciferase assay.


Thanks for any suggestion!

Or just do a large scale alkaline lysis miniprep. Just grow a larger culture and add more volumes of the solutions. To get good quality, it is important to use a phenol-chloroform extraction step (which is not always common in a miniprep).

I've used homemade Guanidine-HCl methods for aspgillus nucleic acid extractions. aspergillie may be familiar with this if I'm inferring correctly from his screen name... I don't think its any cleaner or more efficient than an alkaline lysis prep if you include a phenol extraction. Just another option.

Thanks your guys very much!

My problem is I do not like the phenol-chloroform extraction. I am afraid the DNA will contain the phenol-chloroform remains, which will affect my transfection experiments!

Thanks!

hi
its sure that CsCl2 gradient is the best way to get good quality of DNA though it is complicated but if u try 2/3 times i think u will find it more easier.

another way to get good quality DNA is PEG8000 purification. its very tedious job. but its good and take shorter time than CsCl2 gradient but u can get some time for sleep while u perform cscl2 gradient.

by alkaline lysis method u can get good quality DNA (like sequencing prep) but may be it is not possible to get higher amount of DNA in this mehotd.

I have used the qiagen endotoxin free kit and although sometimes the yield is a bit on the lower side...the qaulity is really good. A couple of years back...i extracted some basic pgl3-luc plasmid using choloroform method and i got a really huge yeild although the quality was really crappy. I did not get any light units after transfection. The qiagen endo-free definitely worked wonders....

I would go for the qiagen maxi prep anyday...

It depends on the coli strain if you need to include a phenol-chloroform extraction step. If it has endonucleases, you have to, otherwise it will cut your DNA when you do a restriction. For other strains it's not really necessary.
The DNA you get out of the "large-scale miniprep" is good enough for sequencing and cloning.

@ Vanussci:
I don't know if guanidine-HCl works with bacteria. Correct me if I'm wrong, but guanidine-HCL extracts the proteoglycans by which the cell wall of the aspergillus becomes weak. And I don't know of the outher cell-wall of the coli contains (enough) proteoglycans to be cut away. There is difference between a proteoglycan and a lipopolysaccharide.

Does anybody know more about this?

i think the Mackerery-Nagel Anion exchange kit gives the best results for a mxiprep..quality as well as quantity...never had a problem ..try it