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 Celsius 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 Celsius for EXACTLY 30 seconds
5. Put it on ice for 2 minutes.
6. The add .5mL (500μL) SOC 
7. Incubate at 37 degrees Celsius 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!

04/18/14

Update on LR's: LR's were left in the incubator for too long and they dried out. Trying to save 4 and 5.

PCR

Reaction Mixture:

• 1uL Template (entry vector eYFP_151ts; ie miRNA 451 target sites)
• Primers (comes in 100uM concentration) corresponding to the gene (2 primers)
• --> we need 2uL of 5uM conc. of each primer.
• --> we have to do 1–>20 dilution.
• 35uL of buffer/enzyme mix (comes in the pack)

Put reaction mixture into Thermocycler:

• Edit Settings
• initial Denature at 95 deg Celsius (for 5 mins)
• Cycles (35 cycles):
  • 95 deg Celsius for 30sec
  • 56 deg Celsius for 30 sec
  • 72 deg Celsius for 30 sec
• Final annealing at 72 deg Celsius for 10 mins
• The hold at 4 deg Celsius indefinitely.

Labeling plates:

• iGEM
• Date
• What they are

04/20/14

Making gel:

• TAE + Agarose powder
• 1% solution (1g/100mL)
• --> Using 50mL, so 0.5g of Agarose
• --> Added 0.496 - 0.506g

Microwave

• --> remember half-open cap!
• --> keep watch
• --> adjust power level (first higher, then lower)
• --> bring up to simmer till
• --> all melted

Once cool enough to touch (~10 mins)

• --> Add SYBR Safe (orange; liquid keep away from light)
  • --> a  dye that causes DNA to fluoresce; VERY important!
  • --> turns it pink
  • --> 10,000x working concentration (so, 5uL to 50mL)

Update on LR: plates didn't make it. Using backup plates

1. 1-2_CMV_mKate
2. 1-2_TRE-t_mKate
   6.  1-2_TRE-t_tagBFP

Picking Colonies:

14mL Round bottomed tubes
LB medium (3mL)
Antibiotic (1000x conc.)
--> important or else other bacteria can grow, and the bacteria may lose their plasmids
--> 3uL to 3mL medium

• Take 2uL pipet and tip
• Scoop up colony (try not to get agar)
• Swirl and pipet up and down (Kyle)
• OR, leave pipet tip there (Katie)

Labeling Tubes:

"1-2_CMV_mKate" (1-2 is the destination vector, CMV is the promoter and mKate is the gene)

Running gel:

Check terminals (runs form +ve to -ve)
Fill with TAE till it covers the agarose gel (can leave the tray in)
Add dye (orange G)
--> 6x conc. so 8uL to 40uL (note, here the volume of dye is large enough to affect total conc.)

1. Ladder
2. 1 (15 uL) (for imaging) (1 = Left, EE)
3. 2 (15 uL) (for imaging) (2 = Right, SS)
4. Space
5. 1 (~30 uL; fill as much as you can) (for extraction)
6. 2 (~30 uL; fill as much as you can) (for extraction)

Wait till about halfway down ~30 mins

Extraction from gel:

1. Image the gel
2. Cut the extraction bands under UV (to see the bands)
3. Follow protocol

04/20/14

Extraction from gel (similar to miniprep):

--> Weight

1. 106mg
2. 96mg

--> Add 3x vol of QG

1. 318uL (1mg of agarose --> 1uL)
2. 288uL

-->put in heat bath to melt (50 deg Celsius for ~10 mins; vortex every 2-3 mins?)
--> 1x vol of Isopropanol (original volume of gel)

1. 106uL
2. 96uL

-->Centrifuge at 13,000 rpm for 1 min; discard flowthrough
--> Add 500uL of QG
--> Centrifuge at 16,000 rpm for 1 min; discard flowthrough
--> Add 750uL of PE
--> Centrifuge again, I assume (not written in notes); dump PE
--> Dry run centrifuge
--> Add 50uL EB (elution buffer), let sit for 1 min (make sure to get it on the filter!)
--> Centrifuge; DO NOT discard flowthrough (this contains the DNA)
--> Nanodrop to measure concentration

Protip on pipets:

Try to work with 1 hand and not move the pipet much. Eg, take something, open with one hand, pipet things in, place it back down on the rack, etc.