Aklaline Lysis Plasmid Isolation (High Throughput)
Fri, Sep 03, 2010, 1:14 pm CDT
A quick and dirty protocol that yields double stranded plasmid template of approximately 3 mg per sample. Colonies are grown in 96 deep well plates (~1 mL sample volume), and then subjected to alkaline lysis.
Keywords: Alkaline Lysis, plasmid preparation, high throughput
Bead Beating DNA Extraction Protocol
Fri, Sep 03, 2010, 1:16 pm CDT
Soils can be a pain in the rear to extract DNA from. We've tried to work this protocol to eliminate as much bias as possible from the final prep. Bead beat too long and you'll ruin the DNA from your Gram negatives. Bead beat too short and you risk not cracking open your Gram positives. To increase final DNA yields, you could also try adding 1 to 2 uL of GlycoBlue with your 95% EtOH precipitation.
Keywords: bacterial DNA isolation, bead beating
Fri, Sep 03, 2010, 1:07 pm CDT
The following PDF is an article that was published by the now defunct Bethesda Research Laboratories back in 1985 (before half the people who read this were even born). It discusses the effects of both incubation time and temperature, as well as centrifugation time. The moral is ... spin your DNA prep down for at least 30 minutes.
**I don't think posting this poses any copyright issues. My apologies if it does.**
Keywords: EtOH precipitation
Acetone/EtOH Bacterial RNA Isolation
Fri, Sep 03, 2010, 1:15 pm CDT
A protocol used in our lab for isolation of RNA from bacterial cultures. This protocol works equally well for Gram positive and Gram negative bacteria (we've done it with both). The acetone/ethanol mixture is great for long term storage of samples prior to RNA isolation. Cells are theoretically permeabilized and proteins (including RNAses) are inactivated.
Keywords: RNA isolation, RNA storage