Team:Caltech/Protocols

Recipe for Agar/LB plate:

1) Fill two bottles with 500 g of nanopure water, 10 g of Tryptone, 10 g of NaCl, 5 g of yeast extract, and 15 g of Agar.

Recipe for 50% Glycerol Stock:
1) Pour 50 mL of Glycerol and 50 mL of pure water into a graduated cylinder.
2) Cover with parafilm and turn over a couple of times to mix.
3) Use the Nalgene vacuum to sterilize the stock.
4) Use the bunsen burner to keep air sterile and close the cap.

Recipe for Enrichment Minimal Media consists of

• 1.0712 g of K2 HPO4
• 0.5239 g of KH2 PO4
• 79 mL of Phosphate solution
• 100 mL of Salt Solution I
• 100 mL of Salt Solution II
• 10 mL of Wolfe's Vitamin Solution
• 1 mL of SL-10 Trace Element Solution.

and then add 5 mg of whatever chemical used in two flasks, one with vitamins and one without.

Transforming DNA Protocol:
1) Thaw competent cells on ice: 15K, 15J, 15O, 15M.
2) Get 10 micro Liters of pure water.
3) Pipette out DNA from the source plate and put it in the appropriate tubes.
4) Flick the competent cells in the tube gently.
5) Pipette 40 micro Liters of competent cells into the DNA.
6) Pipette 2 micro Liters of the DNA and put it in the competent cell tubes.
7) Stir a bit.
8) Leave on ice for 30 minutes.
9) Heat Shock for 45 sec by using a water bath set to 42°C and then thawing on ice for 2 min.
10)Pipette 500 micro Liters of S.O.C. (LB + glucose) into each competent cell tubes.
(STILL CONFUSED ABOUT THIS PROTOCOL NOT SURE IF RIGHT)

Making Plates: 1) Fill the bottom of plate with the LB/Agar solution.
2) Add 100 mg/mL of antibiotics into the plate.
3) Shake gently.

I don't know what this is called...Putting 100 mg soil into each tube, then put the tubes in shakers overnight at 30°C and room temperature respectively???

Starter Competent Cell Culture Recipe?
1) Take the competent cell tubes out of the freezer.
2) Vortex it and take 100 micro Liters from it and pipette it into the labeled plates.
3) Put in some beads and shake gently.
4) Put the dirty beads away in the dirty bead container.