Matt Gethers/CRI, Thailand/Labwork/CRI Biotechnology Lab Specifics

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CRI Biotechnology Lab Specifics

Pipettes and Tips

Fresh tips are located in my zone (bay C, I think). For tips that aren't ruined completely (dyes, caustic chemicals, etc.), place the tips in a container with water. When the container is full, put the tips in the bucket under the sink - these will be autoclaved. For tips I don't need, throw in the trash (no sharps bin).

The 0.5 μl pipetteman is shared between the two benches. Other pipettman are in the individual stations.

Waste Disposal

For pseudomonas waste, be sure to place it in a bag (in a drawer under the gel boxes) to be autoclaved and place it on the waste cart. Mayuree said I should separate plates from agar so the two aren't floating together after autoclaving. I should clarify before I actually do it.

Biohazard Hoods

Use the laminar flow hood (bay A) in instances where I need my samples to remain sterile, but I'm not particularly concerned about my own saftey.

Use the biohazard hood when working with pseudomonas aeruginosa(opprotunitistic pathogen) in bay B. Be sure to clean the hood and pipettes with ethanol before use (like a tissue culture facility). Turn the key to activate the air flow, hit the buttons for the light, bunsen burner, power (next to bunsen burner button), and turn on the gas below the hood.

Chemicals

Located in Bay A, are arranged alphabetically on shelves. There is also a catalog on the door of one of the closets.

Microwaves

Located both in Bay A and Bay C.

Razors

Are located both adjacent to the gel imager in bay C and in the second room (not mine) of bay C on the wall closest to the LB Agar, etc.

Gels

Both 1% and 1.8% agarose are in the incubator on the other side (not my side) of bay B. Combs and the gel box are located in my side of the bay. Guoy says to the loading buffer is 10x, so dilute appropriately. She also says to use 5 μl of λ marker in a lane.

Lare wells on the comb can hold ~15 μl of sample (depending on the thickness of the gel, of course), so plan accordingly.

Ice

Ice is located on the 5th and 6th floors in zone A where the incubators are on the 8th floor. Buckets are located in each zone, and smaller styrofoam containers are located above the sink.

Plates

Located in the other bay of zone C (not mine). Use the 15 cm plates for transformation. Each plate holds 15-20 ml of LB.

Antibiotics and Other Stocks

Reagent Stock Dilution
Ampicillin 100 mg/ml 1/1000
x-gal 2% Pi Koy says 1/104; Pi So says to put 40 μl on a 15 cm plate.
IPTG 200 mM Pi Koy says 1/2000; Pi So says to put 40 μl on a 15 cm plate.
Loading Buffer 10x 1/10

Sequencing

Submit 3 μl of miniprep template per primer to Eh (spelling?) with name, date, and perhaps plasmid type. Good if you can estimate the concentration of DNA, but he can do it as well. If sequencing with a standard primer, there's no need to provide it, otherwise, I may need to do so.

Mass Spec

In wing B. To measure absorbances, click menu (if not there already), select #1 "Photometric." Use the "Get WL" button to set the appropriate wavelength (600 for cultures, 260 for DNA). I need two blanks. Put cuvettes with blank in both ports and hit "auto blank". Then remove the cuvette with blank nearest the keyboard of the spec and replace it with your sample. Be sure to close the door for all readings (blanks included).

To do the Bradford Assay, go to the main menu on the Spec and press "F4" to allow PC control. Click "SP" on the desktop of the computer and hit OK when it prompts you to enter a file location. Right click on the worksheet and click properties and click the box for "Auto Fill". Then click the "connect" button to sync with the Spec and then press "Read Unk" to collect data. To close the program, just x out of the program and go to the main menu on the spec - it will disconnect automatically.