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