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Experiments and Protocol

For MCF tubes:

x = the number of transformations you are doing
Place the SOC medium (from the fridge) in the incubator
Also place X plates in the incubator (With the appropriate antibiotic resistance, Chl for complete, Amp for parts)
Make an ice-water bath
Obtain X MCF tubes and a float
Add 50 ul of competent cells
Add 2ul of the DNA
Place it in the ice-water bath for 2 mins
While waiting, turn on the sink's hot water, obtain a beaker, add hot water, and using a thermometer, adjust water to 42o C (using the hot plate if needed)
Place tubes in the incubator for 30 seconds
Another 2 mins on ice
Add 1 ml of warm SOC medium
Place each tube in the incubator (37o C) for 1 hour
Plating

TENS procedure (make fresh each time)-To 4.5 ml TE solution add 250 ul 10% SDS and 250 ul 2M NaOH
Transfer 1.5ml bacterial culture to a labelled MCF tube
Centrifuge for 30 seconds to pellet bacteria
Pour off most of the growth medium. Leave approximately 100ul in the tube
Resuspend the pellet by shaking, vortexing, or tapping the tube vigorously. Make sure the bacteria are completely resuspended and no clumps remain before continuing.
Add 100ul TENS to the tube
Mix well by inversion for 2 minutes. Do not shake the tube. The solution will get viscous
Add 150ul sodium acetate and mix well
Centrifuge for 3 minutes
Transfer the supernatant (liquid) to a clean, labeled MCF tube
Add 1ml 75% Ethanol to the tube and mix well by inversion. You may see DNA as faint white strands in the liquid. The tube should be nearly full of liquid. Sharpie brand pens have an ink that is soluble in Ethanol. Make sure your labels do not get removed accidentally.
Centrifuge 5 minutes to pellet the DNA. Place the tubes in the centrifuge with the hinges out to make finding the pellet easier
Pour off the 95% Ethanol
Place the tubes upside-down and allow them to dry completely
Resuspend the pellet in 25 uL TE
Use 10 uL of the DNA in a restriction digest or 1 uL for PCR

obtain the plasmid prep tubes (in incubator), new MCF tubes, enzymes in the freezer
For each new tube, prepare the master mix(MM):

Each tube needs 18ul of the MM, in order to get enough MM into each tube. If you have x digest, do x+1 MM. (Ex. If I need to do a digest for one tube, I will prepare 30ul of MM, but still put 18ul into the tube)
Label the new tubes with corresponding tubes and change the "P" into a "D"
Add 3ul DNA and 15ul MM into each tube
Obtain a tube float and put all the tubes into the dry incubator for at least an hour.

Obtain one 125ml conical flask, 1x TAE solution, and agarose
With the balance, measure 0.32g of agarose
Use a measuring cylinder and measure 40 ml of 1x TAE solution
Put the measured powder and liquid into the conical flask (make sure no powder is stuck to the wall)
Use plastic film and wrap around the opening of the flask, BUT remember to leave a hole. Do not completely seal the flask
Microwave the solution for 1 minute
Use a glove and transfer the solution from the Microwave to a black top table
Find the well for the gel, duct tape, and combs (depends on the number of digest you have, you need one or two combs)
Tape the edges of the well and make sure the bottom is sealed. Put the combs in
When the microwaved solution is cooled down a bit, add 4 ul of ethidium bromide into the flask and swirl
Pour the solution into the well and wait til it forms a gel like substance
If you need to store it for future uses, obtain a zip lock bag
Tear off the tape carefully, but leave the combs in
Store them into the refrigerator not the freezer

Team:Gaston Day School/Experiment