BISC209: Lab3: Difference between revisions
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==Lab 3: Continue Soil Microbial Communities & Diversity Project== | ==Lab 3: Continue Soil Microbial Communities & Diversity Project== | ||
'''Protocol: PCR Amplify Genomic DNA from Universal Bacterial Primers'''<br> | '''Protocol: PCR Amplify Genomic DNA from Universal Bacterial Primers'''<br> | ||
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 bacterial population in our study, we will amplify by polymerase chain reaction only the bacterial DNA by using primers that are universal for all 16s rDNA. | |||
Revision as of 18:57, 17 December 2009
Lab 3: Continue Soil Microbial Communities & Diversity Project
Protocol: PCR Amplify Genomic DNA from Universal Bacterial Primers
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 bacterial population in our study, we will amplify by polymerase chain reaction only the bacterial DNA by using primers that are universal for all 16s rDNA.
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?
