BISC209: Lab3

Lab 3: Continue Soil Microbial Communities & Diversity Project

Protocol: PCR Amplify Genomic DNA from Universal Bacterial Primers</font size="+1"
Our genomic DNA isolation has, no doubt, resulted in a mixed DNA population from a myriad of microorganisms as well as, probably, some contaminant DNA from plants, insects, or other life forms in the soil community. Since we are only interested in the scope of our bacterial population in this study, we will amplify by polymerase chain reaction only bacterial DNA by using three "universal" bacterial primers :a forward primers, 27F, and a reverse primer, 1492R. The 16S rDNA sequence is particularly good target gene for amplification because this gene (encoding a ribosomal subunit) contains conserved sequences of DNA common to all bacteria as well as divergent sequences unique to each species of bacteria.

DNA diversity: complete prep for pyrosequencing? OR rDNA library steps: (Use soil DNA from lab 2) DNA library:

Clean and PCR Amplify for rDNA using Irene's universal primers (Irene's notes)- 3 hours

CULTURES: (Primary plates) observe-count, describe, select soil microbes for further culturing (compare anaerobic, aerobic, microaerophillic) - search web for pics of microbes.

techniques: colony morphology, number (see mIcrobial safari= wagner) and problem solve to "discover" 2 isolation methods (serial dilution and pour plates vs streaking for isolation)

Isolation to pure colony step: #1: How will they pick what/where on plate to isolate? (secondary plates) Each student picks different 4-6 colonies and restreaks on same media (4-6 different plates). Incubate room temp.grow 24 hours, move to CR.

Microscopy introduced: select something that looks isolated and stain and compare to stock cultures.

Control stocks for: gram pos, gram neg and capsule, acid fast and endospore

Simple stain Gram stain

What do you learn from this?