Best plasmid miniprep? - (Sep/06/2009 )
I'm screening for colonies with inserts. If I dilute the DNA to 2 ul in 50, I doubt I'll be able to see it on a gel.
Are you suggesting that I follow the miniprep procedure as I've written it, but use KOAc, leave out the PEG portion, and go directly to ethanol precipitation? Or you just suggesting a way to clean up what I already have?
Why is KOAc used instead of NaOAc?
You should easily have enough DNA to visualize bands after the cuts I suggested. A miniprep of 5 ml should yield 3 ug or more of DNA with a high copy plasmid (100 ng/ul with 30 ul). 2 ul of this will have 200 ng of DNA, and 40% of that should have 80 ng, easily detectable with a gel.
If you are getting substantially less DNA from your prep, then you should check the antibiotic in your growth medium and figure out where the DNA is going.
The change to KOAc from NaOAc precipitates the SDS as KDS after lysis, making the isolation of genomic DNA easier. It will form a white precipitate when the lysis is neutralized.
I suggest that you don't need to clean up your existing DNA, but simply to use less of it in higher volume.
You could also clean up your DNA effectively with a phenol/chloroform extraction, but I don't recommend that as a first step without some guidance.
I have been using a new kit from Zymo called the Zyppy Miniprep kit. Its way faster than standard minprep kits because it bypasses the conventional pelleting and resuspension steps. It has really high yields too.. way better than Qiagen. I buy it through Alkali Scientific because they have a good price on it and free shipping.
sciencelover, its pretty clear from your posts that you are spamming with advertising for your company.