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