BISC209: Lab5
LAB 5: Con't. Project: Soil Microbial Communities & Diversity
Preparing your clones for sequencing analysis
When you examine your transformation plates after an overnight incubation, you should see hundreds of identical looking well isolated colonies, all containg the vector which expresses the kanamycin resistance gene and allows the clones to grow on this media with kanamycin that normally would disrupt bacterial protein synthesis and kill the cells. We also know that each of the vectors imparting Kan resistance contains a 16S rDNA insert from one of your soil flora. We know that insert is in the vector because it is responsible for disrupting expression of the ccdB (control of cell death) gene which, if not disrupted, expresses the ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. We hope there are hundreds of 16s rDNA gene fragments from DIFFERENT soil bacteria.
You and your partner will be given a 96 well sterile plate to fill with a bit of each of the clones you want send away for sequencing.
Preparing Glycerol Stocks
1. Streak the original colonies out on LB plates containing 50 μg/ml kanamycin-labeling each with your team color initial, eg. Tues red= TR-1, R-2, R-3, etc up to 96. Incubate at 37C overnight.
2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50 μg/ml
kanamycin. Keep your labeling clear.
3. Grow with shaking to log phase (OD600 = ~0.5)
4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a
cryovial and a well in the 96 well plate. Keep the numbering straight!!! Make a template in your lab notebook.
5. Give the plate to your instructor to seal and send away. Store the remainder at -80°C .
The sequences should come back in a week or two.
ID Cultured Bacteria by 16srDNA Sequencing and Analysis
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