Shinjini Saha

Cloning:

4/13/14:

Done today: L-R reactions.

Protocol:

• 1 μL of everything (μ = 03bc + alt + x in Word)

Conc:

Everything = 5 fmol  (5 fmol per μL)

Destination vector = 10 fmol

• Put 1 μL of everything into the mix. Make sure all liquids coalesce
• Then add 1μL of Clonase
• After everything is added, pipet up and down. Careful not to add air bubbles. Then centrifuge.  If air bubbles form: tap or manual centrifuge
• Incubate overnight at room temperature

Reaction tube numbers:

1. (Kyle) TRE-LacOid_mKate in DEST 1-2
2. (Erik) Hef1a-lacO_ NLS-eYFP in DEST 1-2
3. (Erik) Hef1a-lacO_ eBFP in DEST 1-2
4. (Shinjini) CMV_mKATE in DEST 1-2
5. (Shinjini) CMV_eYFP in DEST 1-2

4/15/14

Transformations:

1. Put competent cells from -80 degree celcius freezer on ice
2. Add 2μL of LR mixture to the cells. Do NOT pipet up and down; swirl instead.
3. Keep cells on ice for 30 minutes.
4. Heat shock cells at 42  degrees celcius for EXACTLY 30 seconds
5. Put it on ice for 2 minutes.
6. The add .5mL (500μL) SOC 
7. Incubate at 37 degrees celcius for 1 hour. (Tape cell tubes together and label!)

Making SOC:

This is the ultra-broth for cell growth, so be extra careful not to get it contaminated.

1. 49mL of SOB
2. 1mL of 20% glucose.

Making Agar:

Follow instruction in pack.

Autoclave for around 20 minutes (+ ~20 minutes to pressurize and depressurize). Cool changing tape!