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Team:UCSF UCB/robert
From 2014.igem.org
Robert's Lab Notebook
WELCOME TO THIS AWESOME MESSY NOTEBOOK
6/9/14 (BootCamp)
PCR for Constitutive Promoters
PCR Reaction:
1 Reaction 4.5X Master Mix
10 ul 5X Phusion Buffer 45 ul
1 ul dNTP's (10uM) 4.5 ul
2.5 ul Foward Primer (10uM) 11.25 ul
2.5 ul Reverse Primer (10uM) 11.25 ul
.3 ul Template DNA 1.35 ul
.5 ul Phusion DNA Polymerase 2.25 ul
332 ul Water 150 ul
Steps
1) Mix Reagents in a 4.5X Master Mix on ice
2) Pipette 50ul of Master Mix into 4 labeled PCR tubes
3) Put into thermocycler for following cycle:
Initial Denaturation 98ºC 30s
35 cycles of
Denaturation 98ºC 10s
Annealing 55ºC 20s
Extension 72ºC 30s
Final Extension 72ºC 5 min
Hold 4ºC FOREVERRRRRR
4) Keep samples for gel extraction
*Forgot to keep 4.5X Master Mix tube on ice *I also made a 1/10ul dilution of each primer when I was supposed to make a 10/100ul dilution
6/10/14
Loading PCR'ed Constitutive Promoters on Gel
50 ul PCR Tube
5 ul 10X Blue Juice Dye
Steps
1) Make a Gel with Cybr Safe
2) Add Blue Juice into PCR tube
3) Load and run gel at 100 volts for 30 min
Gel Photo PCR'ed Constitutive Promoters
Constitutive Promoters pTEF1, m3, m6, m7, m10
6/10/14
Gel Extraction Kit
Steps
1) Cut out DNA from Gel
2) Weigh the slice of gel and and 3X volume of QG Buffer to 1X volume gel. For >2% gels add 6X Buffer.
3) Incubate at 50ºC or 10 mins. Vortex every 2-3 mins.
4) Solution should be yellow. If it is orange or violet add 10ul 3M sodium accetate pH 5 and mix
5) Add 1X gel volume of isoproponal and mix
6) Get a QIAquick spin column (Purple column with cap)
7) Put sample into column and centrifuge for 1 min. Discard flow-through and place column back into tube. for samples >800ul, re-spin the liquid.
8) Add 500ul QG Buffer and spin for 1 min. Discard flow-through and put column back into tube.
9) Add 750ul PE Buffer and spin for 1 min. Discard flow-through and put column back into tube.
10) Do a dry spin for 1 min.
11) Place column into a new 1.5ml microcentrifuge tube.
12) Elute DNA in 50ul (or whatever).
6/11/14
Restricton Enzyme Digest with ApaI
Steps
1) Put all the following into a tube:
40ul DNA
5ul Cutsmart Buffer
0.5ul ApaI
2) Let it digest overnight at room temperature
6/12/14
Restriction Digest with XhoI
Steps
1) Add 0.5ul XhoI
2) Incubate in 37ºC in shaker
3) Run a PCR purification
*DNA concentration: 153ng/ul
DNA Ligation
1 ul 10X Ligase Buffer 0.2 ul DNA backbone 0.2 ul Insert 0.5 ul T4 DNA Ligase 8.1 ul Water
10ul total
-Incubate at room temperature for 2 hours
Transformation
10 ul Ligation 50 ul E.Coli compitant cells
Steps
1) 30 mins on ice
2) 42ºC Heat shock
3) Ice for 2 mins
4) Add 250 ul SOC media and shake/sit at 37ºC for an hour
5) Plate
Re-do Ligation (This time use 0.4ul backbone)
-Also use better competant cells
Re-do Transformation (above)
6/13/14
Yeast Transformation
Steps
Refer to protocal in my binder
6/16/14
Colony PCR for E.Coli
Steps
1) Pick a single colony using a pipette tip and mix in 25ul water in a PCR tube. Do ths for 4-6 colonies on each plate. Save rest of the tube just incase.
2) Set up PCR reaction as below:
**1X Reaction** **6X Reaction**
2X GoTaq Green PCR Master Mix 10ul 60ul
10uM FW Primer 1ul 6ul
10uM RV Primer 1ul 6ul
Water 3ul 18ul
Bacterial cells (Template) 5ul ___ul
*Add 15ul of the 6X Reaction to each tube
Cycles
95 5min
30X: 95 45s
55 30s
72 1min per kb
72 10min
3) Anaylze products on a gel. GoTaq has loading gel in it already. Load 5ul of PCR reaction.
4) For all positive bands, take the rest o the bacterial cells from step 1 and grow them in LB overnight (+antibiotic) for miniprep next day.
6/16/14
Send DNA to be sequenced
-They came back good :)
Colony PCR for Yeast & Patching (Refer to binder for protocol)
Steps
1) Pick 3 colonies and number them
2) Patch colonies onto a plate
3) Mix Yeast cells in 10ul NaOH in PCR tube
4) Boil for 10 min at 95ºC
5) PCR:
95ºC 5min
30X:
95ºC 45s
55ºC 30s
72ºC 1min
72ºC 10min
4ºC FOREVERRRRRRR
Gels
SAG1 ECM18 PRM3 HYM1 ASG7 CLO7 PRM1
| 1KB | 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 |
|---|---|---|---|---|---|---|---|
| ---- | --------- | ---------- | --------- | --------- | -------- | --------- | --- |
| 1KB | 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | 1 2 3 | ||
| ---- | ----- | ------ | --------- | --------- | --------- | --------- |
6/18/14
MiniPrep Constitutive Promoters (Refer to binder for protocol)
Made CBOO8 & CBOO8DB stock dilutions
Glycerol Stocks of CBOO8 & CBOO8DB (Refer to binder for protocol)
6/19/14
Flow Cytometry
Night Before:
1) Start overnight cultures using SD complete media
Day of:
1) Dilute cultures to final ~OD (Optimal Density) in the 96 well shaker plate (overnight cultures should be around ~OD7, so about a 1:100 dilution). Use SD media to make each well a total volume of 1mL
CBOO8
1 2 3 4 5 6 7 8 9 10 11 12
A [------NEG------] [------NEG-------]
B [------PRM2-----] [------PRM1------]
C [------ASG7-----] [------EMC18-----]
D [------PCL2-----] [------PRM3------]
E [------NEG------] [------SAG1------]
F [------CLG1-----]
G [------YDR124W--]
H [------PRM6-----]
in DB008
1 2 3 4 5 6 7 8 9 10 11 12
A [------NEG------] [------NEG-------]
B [------yGEM10---] [------yGEM14----]
C [------yGEM4----] [------yGEM15----]
D [------yGEM5----] [------yGEM16----]
E [------NEG------] [------yGEM6-----]
F [------yGEM11---]
G [------yGEM12---]
H [------yGEM13---]
2) Allow cells to grow on plate shaker for 3 hours at 1000 rpm
3) Induce with Alpha Factor. The Alpha Factor in the freezer is at 3mM and we're using concentrations at 0.1nm, 1nm, 10nm, 100nm, and 1uM
-Concentrations
3mM --> 30ul + 870ul water
⍒
100uM --> 50ul + 450ul water
⍒
10um --> 50ul + 450ul water
⍒
1000nm 50ul + 450ul water
⍒
100nm
*Alpha Factor cannot be refrozen so dump after use
4) Allow induction to proceed on plate shacker for 1.5 - 2 hours
5) Transfer 250 ul of each culture into a 96 V-Well
6) RUn on flow cytometer
-Checklist ✓:
Check status of machine (should be on standyby)
Check fluid boxes and stroage tanks
Do not run plate. Run well.
Have a positive & negative control
Open iGEM account (pw: biobricks)
Make new FACS experiment
-Voltage:
CHeck inspector to see if everything is on
FCS: 250
SSC: 280
FITC: 550
Sample Flow Rate: 1
Sample Volume: 200
Mixing Volume: 100
Mixing Speed: 180
Number of Mixes: 3
Wash Volume: 400
-Wells
Select wells to run (make sure volume is 50ul below actual amount) and there will be a side bar of options on how to adjust your gels (mixing, flow speed)
Run well & save data onto USB
*Don't turn on wifi
7) Analyze Data
6/23/14
Colony PCR (refer to 6/16/14)
Gel result of Yeast Colony PCR:
