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 to send out for sequencing
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 it to 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 on dry ice.
The sequences should come back in a week or two.
ID Cultured Bacteria by 16srDNA Sequencing and Analysis
Besides working on getting your soil sample DNA isolated, amplified by pcr, inserted in a cloning vector, transformed into E. coli, and the 16s rDNA insert sequenced in an automatic sequencer so you can identify a more represenative scope of the bacterial flora in your soil sample, you have been working simultaneously, through traditional microbiological culturing techniques, to isolate and identify some of the culturable bacteria by morphology and metabolism differentiation. Look how much you have accomplished in these few short weeks!!
By this point you have isolated pure colonies of some soil bacteria on general and enrichment media and you have gotten some preliminary information about the morphologic and metabolic characteristic of the bacteria you have chosen to identify. You will continue learning about how these bacteria are different from one another and how they contribute to their community. At the same time we want to identify these bacteria by their 16s rDNA unique sequences in a similar way as we did for our unknown general soil sample bacteria. This time we will use a Taq polymerase that will not be as accurate as our proof reading polymerase we used previously, but should be good enough to get our identifications.
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Prepare DNA from 4 Bacterial Colonies
1. Each person will choose 4 unique colonies (try to pick colonies that are clearly different from each other from a variety of the enrichment media)
2. Touch a colony with a P10 tip and resuspend the non-visible material in 20 microliters of sterile water with 0.05% Non-idet P40 (NP40). NP40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes.
Resist the urge to pick up too much cell material!! The tinest invisible bit will do and is better than too much, which can inhibit the pcr reaction!
3. Repeat for your other 3 colonies.
4. Boil the samples in the 0,05% NP40 for 5 min. You can do this in the thermal cycler if you set a program to boil. This will lyse the cells and to inactivate bacterial enzymes.
PCR AMPLIFICATION of 16s rDNA from lysates
Note: All reagents for the pcr should be kept on ice and the master mix should be thawed on ice. Since Taq can function at room temp, we don't want the reaction to start until all the tubes are in the thermal cycler.
The components below have been aliquoted and prepared for you and are in pcr tubes of your team color. Label the tubes carefully with a Sharpie on the top and side of the tube with a unique identifier for each bacterial colony (your initials and a number: Carl Woese CW-1) and make a template key in your lab notebook as to where in the thermal cycler you put your tubes.
Master Mix recipe for each reaction: TOTAL VOL 23 microliters
WEAR GLOVES AT ALL TIMES AND DON'T TOUCH THE INSIDE OF THE TUBE CAPS OR YOUR PIPET TIPS--Always use a new tip when going into anything in a pcr reaction. Contamination is a real problem in pcr
REAGENT and VOLUME
Promega Master Mix 2x 12.5 microliters
16S_ 8F(15 pmol) primer 2.0 microliter
16S_149R (15 pmol) 2.0 microliter
nuclease free water 6.5 microliter
Add 2 microliters of our boiled lysate with the template DNA
