"
Team:UCSF UCB/derrick
From 2014.igem.org
Derrick's Lab Notebook
5/28/14
PCR of Yeast Promoters
Materials:
1X 4.5x
5x HF Phusion Buffer 10 µl 45 µl
10mM dNTPs 1 µl 4.5 µl
10mM FW Primer 2.5 µl 11.25 µl
10mM REV Primer 2.5 µl 11.25 µl
Phusion Polymerase 0.5 µl 2.25 µl
Template DNA 0.5 µl 2.25 µl
ddH20 33 µl 148.5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Notes:
- from yeast genomic DNA
- 13 different promoters:
- 1KB: YDR124W, SAG1, PCL2, HYM1, FUS3
- 700BP: CLG1, PRM2
- 500BP: PRM6, PRM3, PRM1, FUS2, ECM18, ASG7
Promoters
1) PRM2
2) ASG7
3) FUS2
4) PCL2
5) FUS3
6) CLG1
7) YDR124W
8) HYM1
9) PRM6
10) PRM1
11) ECM18
12) PRM3
13) SAG1
numbering system for promoters
5/29/14
Gel:
Transformation of pHY4:
- used 0.2 µl of pHY4
- sat on ice for 10 minutes before heat shock
- incubate at 37°C for 30 minutes
Gel Extraction of Alpha Inducible Promoters:
- Qiagen Gel Extraction Protocol used
Qiagen column was not used, but protocol was used. may have yielded less efficency
Promoters ng/µl 1) PRM2 13.49 2) ASG7 12.50 3) FUS2 n/a 4) PCL2 9.489 5) FUS3 11.77 6) CLG1 16.30 7) YDR124W 35.75 8) HYM1 23.61 9) PRM6 79.03 10) PRM1 n/a 11) ECM18 14.17 12) PRM3 n/a 13) SAG1 26.07
5/30/14
Miniprep of pHY4
- centrifuge at 4000 RPM
- followed Qiagen protocol
- Concentrations: ng/µl
- 51.15
- 65.40
- 52.89
- 31.65
Digestion of promoters
- Promoters
- 29 µl of PCR product
- 3.3 µl of 10x Cutsmart Buffer
1) Add 0.5 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 0.5 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
3) PCR Purification
Digestion of pHY4
- pHY4
- 43 µl of PCR product
- 5 µl of 10x Cutsmart Buffer
1) Add 1 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 1 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
3) Gel Extraction
Concentrations from PCR Purification:
1) 14.54
2) 4.326 redo, 5.4890
4) -0.05256 redo, 1.730
5) 1.886 redo, 2.642
6) 41.05
7) 3.187
8) 19.09
9) 9.677
11) 1.868
13) 23.78
Concentrations from Gel Extraction
1) 7.749 -pHY4 1
2) 14.87 -pHY4 2
Gel:
6/2/14
ligated #1,2,5,6,7,8,9,13 with the 6kb pHY4 backbone
- only had enough backbone for 8 reactions so chose the 8 promoters with the highest concentration
used Gibson ligation calculator to calculate mLs of reagents of our reaction
Protocol: See ligation protocol
PCR of Yeast Alpha Inducible Promoters 3,4,7,10,11,12
Materials:
1X 4.5x
5x HF Phusion Buffer 10 µl 45 µl
10mM dNTPs 1 µl 4.5 µl
10mM FW Primer 2.5 µl 11.25 µl
10mM REV Primer 2.5 µl 11.25 µl
Phusion Polymerase 0.5 µl 2.25 µl
Template DNA 0.5 µl 2.25 µl
ddH20 32.5 µl 146.25 µl
DSMO 0.5 µl 2.25 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Gel:
Gel Extraction:
- follow Qiagen protocol
- Concentrations (ng/µl):
- 4) 84.15
- 7) 134.3
- 10) 84.14
- 11) 91.99
- 12) 64.64
6/3/14
Miniprep of pHY4
- follow Qiagen protocol
- low yields of plasmid previously due to PE Buffer not containing ethanol
- Concentrations (ng/µl):
- 360.2
- 398.8
- 322.2
- 240.2
pHY4 + Promoters:
- did not grow very well after ligation
- 7,9,13 grew some colonies
- 1,2,5,6,8,9,13 will be re-PCRed
Digestion:
Digestion of promoters
- Promoters
- 50 µl of PCR product
- 5 µl of 10x Cutsmart Buffer
1) Add 1 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 1 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
3) PCR Purification
Digestion of pHY4
- pHY4
- 50 µl of PCR product
- 5 µl of 10x Cutsmart Buffer
1) Add 1 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 2 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
3) Gel Extraction
Gel:
- gel extraction was not performed b/c DNA was chewed up, no clear cut
PCR Purification Concentrations (ng/µl):
- follow Qiagen Protocol
4) 165.1
7) 146.8
11) 68.84
12) 76.15
6/4/13
Digestion of pHY4
- pHY4 was redigested since the first two attempts were unsuccessful
Digestion of pHY4
- pHY4
- 40 µl of PCR product
- 5 µl of 10x Cutsmart Buffer
1) Add 1 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 2 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
3) Gel Extraction
Miniprep of pHY4
- pHY4 was cultured last night, more needed for ligation w/ promoters
- follow Qiagen protocol
- Concentrations (ng/ul):
- pHY4-1: 178.8
- pHY4-2: 155.6
- pHY4-4: 231.7
Gel:
Digestion of pHY4 +positive control
- positive control from Hyun's stock plasmid
Digestion of positive control
- pHY4
- 5 µl of PCR product
- 2.5 µl of 10x Cutsmart Buffer
- 16.5 µl H20
1) Add 1 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 1 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
Digestion of pHY4
- pHY4
- 40 µl of PCR product
- 5 µl of 10x Cutsmart Buffer
1) Add 1 µl of ApaI. Mix gently, spin down, incubate at room temp. for 2 hours
2) Add 2 µl of XhoI. Mix gently, spin down, incubate at 37ºC for 1 hour
3) Gel Extraction
6/5/14
Gel:
Gel Extraction
- follow Qiagen gel extraction protocol
- concentrations (ng/µl)
- + control: 33.23
- pHY4-1: 50.33
- pHY4-3: 45.38
- pHY4-4: 56.06
Ligation of Promoters w/pHY4
0.2 µl of backbone
_ µl of insert
1 µl of T4 ligase buffer
0.5 µl
_ µl of H20
---------------
10 µl total
- sit at room temp for 2 hours
- 1,2,4,8,10,11,12: 8.1 µl H20 + 0.2 µl insert
- 6: 8.0 µl H20 + 0.3 µl insert
- negative control: 8 µl H20 + no insert
transform into 50 µl dH5
Yeast Transformation of 7,9,13
6/6/14
Digestion of pHY4 -negative control had around 29 colonies -redigesting plasmids of control, pHY4 1,3,4 -follow same digestion protocol -note: 50µl of DNA
Colony PCR of promoters: 1,2,4,6,10,11,12
Materials: 1X 8x
GoTAQ Mix 12.5 µl
10mM FW Primer 1.25 µl
10mM REV Primer 1.25 µl
Template DNA 5 µl
ddH20 5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Gel 1:
Gel 2:
Miniprep of Ligation cultures
-miniprep protocol
-used vacuum manifold
-ethanol not completely eluted, may affect sequencing
- Concentrations (ng/ul):
- 1-4: 142.4
- 1-5: 203.7
- 2-4: 317.3
- 2-5: 205.9
- 4-4: 321.5
- 4-5: 284.9
- 6-4: 192.9
- 6-5: 432.9
- 10-2: 103.8
- 10-3: 203.2
- 11-4: 144.7
- 11-5: 226.8
- 12-4: 267.7
- 12-5: 267.7
6/10/14
PCR of Gibson Homology of rtTA to promoters
Materials: 1X
5x HF Phusion Buffer 10 µl
10mM dNTPs 1 µl
10mM FW Primer 2.5 µl
10mM REV Primer 2.5 µl
Phusion Polymerase 0.5 µl
Template DNA 0.5 µl
ddH20 33 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
PCR Purification -follow PCR purification protocol
- Concentrations (ng/ul): 1.PRM2: 190.9 4.PCL2: 149.1 6.CLG1: 412.1 7.YDR124W: 90.37 8.HYM1: 248.9 9.PRM6: 145.7 10.PRM1: 71.88 11.ECM18: 199.7 13.SAG1: 65.55
Gel:
6/11/14
PCR of Gibson Homology of rtTA to promoters
Materials: 1X
5x HF Phusion Buffer 10 µl
10mM dNTPs 1 µl
10mM FW Primer 2.5 µl
10mM REV Primer 2.5 µl
Phusion Polymerase 0.5 µl
Template DNA 0.5 µl
ddH20 33 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
PCR Purification -follow PCR purification protocol
- Concentrations (ng/ul): 2.ASG7: 179.6 12.PRM3: 236.6
Gel
6/12/14
Seamless Cloning of rtTA + promoters & pHY4
-follow seamless cloning procedure
Promoters
1) PRM2
2) ASG7
4) PCL2
6) CLG1
7) YDR124W
8) HYM1
9) PRM6
10) PRM1
11) ECM18
12) PRM3
13) SAG1
6/16/14
Colony PCR of rtTA -transformations of SAG1& PRM1 didn't grow -inoculate colony in 25µl H20 Materials: 1X 4x
GoTAQ Mix 12.5 µl
10mM FW Primer 1.25 µl
10mM REV Primer 1.25 µl
Template DNA 5 µl
ddH20 5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Gel:
Colony PCR pf yeast transformations (promoter+GFP) Materials: 1X 70x
GoTAQ Mix 12.5 µl
10mM FW Primer 1.25 µl
10mM REV Primer 1.25 µl
Template DNA 5 µl
ddH20 5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Gel 1:
Gel 2:
Gel 3:
6/17/14
Colony PCR of Yeast transformations (promoter +GFP) -previous colony PCR failed, need to redo Materials: 1X 80x
GoTAQ Mix 12.5 µl
10mM FW Primer 1.25 µl
10mM REV Primer 1.25 µl
Template DNA 5 µl
ddH20 5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Miniprep of rtTA + promoter Concentrations (ng/ul): ECM18+rtTA #1 308.3 ng/ul ECM18+rtTA #2 231.6 ng/ul PCL2+rtTA #1 162.7 ng/ul PCL2+rtTA #2 247.3 ng/ul PRM6+rtTA #1 248.1 ng/ul PRM6+rtTA #2 327.9 ng/ul ASG7+rtTA #1 351.7 ng/ul ASG7+rtTA #2 593.5 ng/ul HYM1+rtTA #1 221.7 ng/ul HYM1+rtTA #2 413.0 ng/ul PRM2+rtTA #1 324.3 ng/ul PRM2+rtTA #2 339.6 ng/ul PRM3+rtTA #1 351.5 ng/ul PRM3+rtTA #2 316.7 ng/ul CLG1+rtTA #1 289.0 ng/ul CLG1+rtTA #2 276.8 ng/ul YDR124W+rtTA #1 366.0 ng/ul YDR124W+rtTA #2 239.3 ng/ul
6/18/14
Yeast Transformations -two strains (CB008) & (CB008DB) 9 rtTA Promoters: PRM2 ASG7 PCL2 CLG1 YDR124W PRM6 PRM1 ECM18 PRM3 SAG1 GFP + Promoter: HYM1 PRM3 (only DB)
6/19/14
Flow Cytometry of AFRP + GFP
Overnight cultures of CB008+AFRP+GFP diluted ~100x (to a final concentration of OD600 0.5-0.1, Saturated overnight cultures should be OD600 of ~7) in SD complete media, and grown for 3 hours, 1000rpm shaker, 30ºC. Growing in 2mL well plates
Induce with Alpha-factor. Stock is in 3mM. Final concentrations are 0, 1nM, 10nM, 100nM, 1000nM. Alpha-factor cannot be refrozen, so throw leftover away.
Induce for 90mins, but no longer than 120mins
Transfer 250u of each well into a V-bottom 96-well plate containing 10ul of the fixing chemical Cyclohexamide. (thats 4 ul for every 100ul of culture) Cyclohexamide stops protein production by inhibiting ribosomes.
Run on the flow cytometer.
Parameters of flow
FSC: 250
SSC: 280
FITC(GFP): 550
B(RFP): 650
Flow rate: 1µL/sec
Sample Volume: 200µL
Mixing Volume: 100µL
Mixing speed: 180µL/sec
Plate map
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-----]
Notes: -CLG1 x2 Alpha Factor -10ul culture diluted in 1ml of SD Complete
6/20/14
Minipreps of Yeast Colonies (rtTA + promoter) -send for sequencing since previous sequencing failed. colony PCR yielded false positives -follow Miniprep protocol Concentrations (ng/ul): YDR124W.3 241.7 ng/ul YDR124W.4 386.5 ng/ul YDR124W.5 439.6 ng/ul ASG7.3 207.7 ng/ul ASG7.4 319.8 ng/ul ASG7.5 287.2 ng/ul ASG7.6 300.2 ng/ul ASG7.7 338.5 ng/ul CLG1.3 408.0 ng/ul CLG1.4 306.0 ng/ul CLG1.5 277.6 ng/ul CLG1.6 249.8 ng/ul CLG1.7 277.1 ng/ul HYM1.3 440.1 ng/ul HYM1.4 342.4 ng/ul HYM1.5 388.7 ng/ul HYM1.6 226.7 ng/ul HYM1.7 351.2 ng/ul ECM18.3 377.0 ng/ul ECM18.4 426.8 ng/ul ECM18.5 368.8 ng/ul ECM18.6 459.7 ng/ul ECM18.7 372.9 ng/ul PRM3.3 430.4 ng/ul PRM3.4 441.8 ng/ul PRM3.5 361.7 ng/ul PRM3.6 375.6 ng/ul PRM3.7 333.1 ng/ul
6/23/14
Digestion of GFP from pHY4 + promoters -sequencing failed fpr nearly all promoters -GFP possibly not cut all the way -sequencing shows that GFP not fully digested
Already cut plasmid 49ul
plasmid 5ul
Cutsmart 6ul
Not1 0.5ul
Xho1 1ul
incubate at 37ºC for 2 hours
Gel Extraction Concentrations (ng/ul): ASG7 23.77 ng/ul CLG1 16.59 ng/ul HMY1 12.96 ng/ul ECM18 21.39 ng/ul PRM3 15.20 ng/ul -done by Ianto & Jessica
Concentrations (ng/ul):
YDR124W 15.46 ng/ul
SAG1 12.10 ng/ul
PRM6 21.74 ng/ul
PRM1 4.186 ng/ul
PCL2 6.348 ng/ul
PRM2 0.451 ng/ul
Gel:
6/24/14
PCR of rtTA & promoters -previously only been amplifying half of rtTA -reorderedplasmids to include activation domain of rtTA
Materials: 1X
5x HF Phusion Buffer 10 µl
10mM dNTPs 1 µl
10mM FW Primer 2.5 µl
10mM REV Primer 2.5 µl
Phusion Polymerase 0.5 µl
Template DNA 0.5 µl
ddH20 33 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
6/26/14
- Flow cytometry, testing (triplicate) PRM2, ASG7, PLC2, CLG1
- Alpha-factor concentrations: (Induce for 90mins)
- (0nm, 0.5nm, 1nm, 10nm, 100nm, 1000nm, 3000nm)
Starting concentration is 3mM or 3,000,000nM Make 100x stocks and add 10ul to the 1mL cultures: 0nM 50nM 100nM 1000nM 10,000nM 100,000nM 300,000nM
Plate 1 & 2 Alpha Factor Concentration Map:
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
Plate 1:
H G F E D C B A
1 [--------CB008-1--------]
2 [--------CB008-2--------]
3 [--------CB008-3--------]
4 [--------PRM2-1---------]
5 [--------PRM2-2---------]
6 [--------PRM2-3---------]
7 [--------ASG7-1---------]
8 [--------ASG7-2---------]
9 [--------ASG7-3---------]
10 [--------PCL2-1---------]
11 [--------PCL2-2---------]
12 [--------PCL2-3---------]
Plate 2:
H G F E D C B A
1 [--------CLG1-1---------]
2 [--------CLG1-2---------]
3 [--------CLG1-3---------]
4
5
6
7
8
9
10
11
12
6/27/14
Continuation of CB008+AFRP+GFP FACs.
PRM6+GFP did not grow up yesterday.
ROUND 3 PLATE 1 PROMOTER MAP
H G F E D C B A
1 [--------CB008-1--------]
2 [--------CB008-2--------]
3 [--------CB008-3--------]
4 [--------YDR124W-1------]
5 [--------YDR124W-2------]
6 [--------YDR124W-3------]
7 [--------PRM3-1---------]
8 [--------PRM3-2---------]
9 [--------PRM3-3---------]
10 [--------PRM1-1---------]
11 [--------PRM1-2---------]
12 [--------PRM1-3---------]
ROUND 2 PLATE 2 PROMOTER MAP [DID NOT COMPLETE]
H G F E D C B A
1 [--------ECM18-1--------]
2 [--------ECM18-2--------]
3 [--------ECM18-3--------]
4 [--------SAG1-1---------]
5 [--------SAG1-2---------]
6 [--------SAG1-3---------]
7
8
9
10
11
12
Instructions for starting flow:
- Turn on and wait 30mins for the machine to warm up
- Re-initialize the HTS, them prime it three times. You'll get a bubble in the lines if you don't
- Run CST!!! Follow the sheet in front of the monitor for more instructions for that part. (CST uses beads to calibrate the laser detection.) add one drop into 250ul of sheath fluid in A1.
- Bead LOT ID: use the one that is most current
- Load A1-A4 with bleach and B1-B4 with water [flip for the opposite corner] -Cytometer>CST. Make sure cytometer performance results passed
Run Clean Plate: 1. Click on experiment. Experiment>Open experiment 2. Open clean plate "Daily Clean" - 96 well U-bottom" 3. Be here to see if the cleaning is going correctly, low events (less than 100events/sec for bleach and less than 10events/sec for water) 4. If over the events, run another clean plate 5. View events in aquisition dashboard in view>Acquisition dashboard 6. Can also make a clean plate using HTS>Clean
6/14/30
Continuation of CB008+AFRP+GFP FACS. Also testing Constitutive pTEF1 promoters +GFP today
ROUND 4 MAP
H G F E D C B A
1 [--------ECM18-1--------]
2 [--------ECM18-2--------]
3 [--------ECM18-3--------]
4 [--------SAG1-1---------]
5 [--------SAG1-2---------]
6 [--------SAG1-3---------]
7 [--------CB008-1--------]
8 [--------CB008-2--------]
9 [--------CB008-3--------]
10
11
12
Constitutive Promoter MAP
1 2 3 4 5 6 7 8 9 10 11 12
A [-CB008-]
B [-pTEF1-]
C [-m3----]
D [-m6----]
E [-m7----]
F [-m10---]
G
H
7/2/14
Flow of AFRP +GFP CB008 -redoing since data from previous FACS run had some odd behaviors and high standard error
Plate 1:
H G F E D C B A
1 [--------CB008-1--------]
2 [--------CB008-2--------]
3 [--------CB008-3--------]
4 [--------PRM2-1---------]
5 [--------PRM2-2---------]
6 [--------PRM2-3---------]
7 [--------ASG7-1---------]
8 [--------ASG7-2---------]
9 [--------ASG7-3---------]
10 [--------PCL2-1---------]
11 [--------PCL2-2---------]
12 [--------PCL2-3---------]
Plate 2:
H G F E D C B A
1 [--------CLG1-1---------]
2 [--------CLG1-2---------]
3 [--------CLG1-3---------]
4 [--------YDR124W-1------]
5 [--------YDR124W-2------]
6 [--------YDR124W-3------]
7 [--------PRM6-1---------]
8 [--------PRM6-2---------]
9 [--------PRM6-3---------]
10
11
12
Alpha Factor Concentrations:
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{3000 nM}----------------]
B [-------------------{1000 nM}----------------]
C [-------------------{100 nM}-----------------]
D [-------------------{10 nM}------------------]
E [-------------------{1 nM}-------------------]
F [-------------------{0.5 nM}-----------------]
G [-------------------{0 nM}-------------------]
H
-CB008-1, PRM2-1-3, CLG1-1-3 didn't grow well, cultures very dilute and clear. added 20µl of cells instead of 10 -plate 1&2 follow same concentrations
7/7/14
Flow of AFRP +GFP CB008 & pTEF1 + Mutants + GFP
Plate 1:
H G F E D C B A
1 [--------CB008-1--------]
2 [--------CB008-2--------]
3 [--------CB008-3--------]
4 [--------PRM1-1---------]
5 [--------PRM1-2---------]
6 [--------PRM1-3---------]
7 [--------ECM18-1--------]
8 [--------ECM18-2--------]
9 [--------ECM18-3--------]
10 [--------PRM3-1---------]
11 [--------PRM3-2---------]
12 [--------PRM3-3---------]
Plate 2:
H G F E D C B A
1 [--------SAG1-1---------]
2 [--------SAG1-2---------]
3 [--------SAG1-3---------]
4 [--------AGA1-1---------]
5 [--------AGA1-2---------]
6 [--------AGA1-3---------]
7 [--------PRM2-1---------]
8 [--------PRM2-2---------]
9 [--------PRM2-3---------]
10 [--------CLG1-1---------]
11 [--------CLG1-2---------]
12 [--------CLG1-3---------]
Alpha Factor Concentrations:
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{3000 nM}----------------]
B [-------------------{1000 nM}----------------]
C [-------------------{100 nM}-----------------]
D [-------------------{10 nM}------------------]
E [-------------------{1 nM}-------------------]
F [-------------------{0.5 nM}-----------------]
G [-------------------{0 nM}-------------------]
H
Plate 3:
1 2 3 4 5 6 7 8 9 10 11 12
A [-CB008-]
B [-pTEF1-]
C [-m3----]
D [-m6----]
E [-m7----]
F [-m10---]
G
H
-PRM2 had an OD600 of 0.3, added 100µl to 1ml of media
7/8/14
Colony PCR of pTET + GFP in CB008 & CB008DB -leu2 integration site -primers RA151 FW & RA 145 REV -boiled colony in 5µl 20 mM NaOH for 20min @ 95°C
Materials: 1X
GoTAQ Mix 10 µl
10mM FW Primer 1 µl
10mM REV Primer 1 µl
Template DNA 3 µl
ddH20 5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
-no colonies worked for the PCR
7/9/14
-Jeffrey previously cloned all constiutive promoters & rtTA (except for m3) & digested w/ PME1 -Eric & I transformed this into yeast CB008 & CB008DB containg pTET + GFP in the leu integration site -consti. & rtTA went into the ura site -follow yeast transformation protocol
7/10/14
Flow of CB008DB+AFRP+GFP -testing in strain CB008DB -strain has no Bar1 which degrades alpha factor -CB008 has Bar1 -hope to reach plateau in flow analysis
Plate 1:
H G F E D C B A
1 [--------CB008DB-1------]
2 [--------CB008DB-2------]
3 [--------CB008DB-3------]
4 [--------CLG1-1---------]
5 [--------CLG1-2---------]
6 [--------CLG1-3---------]
7 [--------PRM2-1---------]
8 [--------PRM2-2---------]
9 [--------PRM2-3---------]
10 [--------PCL2-1---------]
11 [--------PCL2-2---------]
12 [--------PCL2-3---------]
Plate 2:
H G F E D C B A
1 [--------SAG1-1---------]
2 [--------SAG1-2---------]
3 [--------SAG1-3---------]
4 [--------AGA1-1---------]
5 [--------AGA1-2---------]
6 [--------AGA1-3---------]
7
8
9
10
11
12
Alpha-Factor Concentrations
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
Miniprep of PTS47 -PTS47 pNH406 + rtTA -11E3178 pTET-MFAlpha -follow Miniprep protocol
Concentrations (ng/ul): PTS47 pNH406 + rtTA: #1 637.7 #2 387.7 11E3178 pTET-MFAlpha: 423.0
7/11/14
Flow Cytometry with CB008DB+AFRP+GFP
H G F E D C B A
1 [-------CB008DB-1-------]
2 [-------CB008DB-2-------]
3 [-------CB008DB-3-------]
4 [---------PRM1-1--------]
5 [---------PRM1-2--------]
6 [---------PRM1-3--------]
7 [--------ECM18-1--------]
8 [--------ECM18-2--------]
9 [--------ECM18-3--------]
10
11
12
Alpha-Factor Concentrations
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
Glycerol stocks of YDR124W, PRM6, & ASG7 did not grow in SD complete media overnight
7/14/14
Flow Cytometry with CB008DB+AFRP+GFP
Plate 1:
H G F E D C B A
1 [--------CB008DB-1------]
2 [--------CB008DB-2------]
3 [--------CB008DB-3------]
4 [--------ASG7-1---------]
5 [--------ASG7-2---------]
6 [--------ASG7-3---------]
7 [--------YDR124W-1------]
8 [--------YDR124W-2------]
9 [--------YDR124W-3------]
10 [--------PRM6-1---------]
11 [--------PRM6-2---------]
12 [--------PRM6-3---------]
Alpha-Factor Concentrations
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
- E3 & F3 had some weird clumping on the bottom of the well and can't be resuspended
- A2 skipped, human error.
7/16/14
[Doxycycline]: 100 mg/ml stock or 100,000 ug/ml (in 100ul Aliquots) Induction time: 6hrs Concentrations needed for 100x:
0 ug/ml
3 ug/ml
6 ug/ml
9 ug/ml
30 ug/ml
60 ug/ml
90 ug/ml
300 ug/ml
600 ug/ml
900 ug/ml
3000 ug/ml
6000 ug/ml
Dilutions by Ianto:
Stock: 100mg/mL
**1x** **100x** Prep
60µg/mL A: 6mg/mL (6:100) 60µL of Stock in 940µL of water
30µg/mL B: 3mg/mL 30µL of Stock in 970µL of water
9µg/mL C: 900µg/mL 150µL of A in 850µL of water
6µg/mL D: 600µg/mL 100µL of A in 900µL of water
3µg/mL E: 300µg/mL 50µL of A in 950µL of water Can also do 100ul of B in 900ul of water
0.9µg/mL F: 90µg/mL 100µL of C in 900µL of water
0.6µg/mL G: 60µg/mL 100µL of D in 900µL of water
0.3µg/mL H: 30µg/mL 100µL of E in 900µL of water
0.09µg/mL I: 9µg/mL 100µL of F in 900µL of water
0.06µg/mL J: 6µg/mL 100µL of G in 900µL of water
0.03µg/mL K: 3µg/mL 100µL of H in 900µL of water
Doxycycline concentrations plate map:
H G F E D C B A
1 [----0 µg/ml----]
2 [---0.03 µg/ml--]
3 [---0.06 µg/ml--]
4 [---0.09 µg/ml--]
5 [---0.3 µg/ml---]
6 [---0.6 µg/ml---]
7 [---0.9 µg/ml---]
8 [---3.0 µg/ml---]
9 [---6.0 µg/ml---]
10 [---9.0 µg/ml---]
11 [---30 µg/ml----]
12 [---60 µg/ml----]
pTEF1 promoters
Plate 1:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008 Control]--------------]
B [---------------[CB008 pTET_GFP Control]-----]
C [---------------[CB008 pTET_GFP pTEF1_rtTA]--]
D [---------------[CB008 pTET_GFP pTEF1 m6]----]
E [---------------[CB008 pTET_GFP pTEF1 m7]----]
F [---------------[CB008 pTET_GFP pTEF1 m10]---]
G
H
Plate 2:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008DB Control]------------]
B [---------------[CB008DB pTET_GFP Control]---]
C [---------------[CB008DB pTET_GFP pTEF1 1of2 ]
D [---------------[CB008DB pTET_GFP pTEF1 2of2 ]
E [---------------[CB008DB pTET_GFP pTEF1 m6]--]
F [---------------[CB008DB pTET_GFP pTEF1 m7]--]
G
H
7/22/14
Plate map
Flow Cytometry with CB008DB+AFRP+GFP
Plate 1:
H G F E D C B A
1 pTEF1-1 [--------CB008DB-1------]
2 pTEF1-2 [--------CB008DB-2------]
3 pTEF1-3 [--------CB008DB-3------]
4 M7-1 [--------ASG7-1---------]
5 M7-2 [--------ASG7-2---------]
6 M7-3 [--------ASG7-3---------]
7 [--------YDR124W-1------]
8 [--------YDR124W-2------]
9 [--------YDR124W-3------]
10 [--------PRM6-1---------]
11 [--------PRM6-2---------]
12 [--------PRM6-3---------]
Plate 2:
H G F E D C B A
1 [--------AGA1-1---------]
2 [--------AGA1-2---------]
3 [--------AGA1-3---------]
4 [--------CLG1-1---------] did not grow as much as the others
5 [--------CLG1-2---------] 20ul instead of 10ul for CLG1
6 [--------CLG1-3---------]
7 [--------PRM2-1---------]
8 [--------PRM2-2---------]
9 [--------PRM2-3---------]
10 [--------PCL2-1---------]
11 [--------PCL2-2---------]
12 [--------PCL2-3---------]
Plate 3:
H G F E D C B A
1 [--------ECM18-1--------]
2 [--------ECM18-2--------]
3 [--------ECM18-3--------]
4 [--------SAG1-1---------]
5 [--------SAG1-2---------]
6 [--------SAG1-3---------]
7
8 there is no PRM3 made, in glycerol stock
9
10 [--------PRM1-1---------]
11 [--------PRM1-2---------]
12 [--------PRM1-3---------]
Alpha-Factor Concentrations
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
7/29/14
Flow Cytometry of Consti+rtTA+pTET+GFP CB008DB Dox run
-All plates are identical
Plate 1:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008DB]--------------------]
B [---------------[CB008DB pTET_GFP]-----------]
C [---------------[CB008DB pTET_GFP pTEF1]-----]
D [---------------[CB008DB pTET_GFP pTEF1 m3]--]
E [---------------[CB008DB pTET_GFP pTEF1 m6]--]
F [---------------[CB008DB pTET_GFP pTEF1 m7]--]
G [---------------[CB008DB pTET_GFP pTEF1 m10]-]
H
Plate 2:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008DB]--------------------]
B [---------------[CB008DB pTET_GFP]-----------]
C [---------------[CB008DB pTET_GFP pTEF1]-----]
D [---------------[CB008DB pTET_GFP pTEF1 m3]--]
E [---------------[CB008DB pTET_GFP pTEF1 m6]--]
F [---------------[CB008DB pTET_GFP pTEF1 m7]--]
G [---------------[CB008DB pTET_GFP pTEF1 m10]-]
H
Plate 3:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008DB]--------------------]
B [---------------[CB008DB pTET_GFP]-----------]
C [---------------[CB008DB pTET_GFP pTEF1]-----] entire lane 10 is induced
D [---------------[CB008DB pTET_GFP pTEF1 m3]--] with wrong concentration.
E [---------------[CB008DB pTET_GFP pTEF1 m6]--]
F [---------------[CB008DB pTET_GFP pTEF1 m7]--]
G [---------------[CB008DB pTET_GFP pTEF1 m10]-]
H
Doxycycline concentrations plate map:
H G F E D C B A
1 [----0 µg/ml----]
2 [---0.03 µg/ml--]
3 [---0.06 µg/ml--]
4 [---0.09 µg/ml--]
5 [---0.3 µg/ml---]
6 [---0.6 µg/ml---]
7 [---0.9 µg/ml---]
8 [---3.0 µg/ml---]
9 [---6.0 µg/ml---]
10 [---9.0 µg/ml---]
11 [---30 µg/ml----]
12 [---60 µg/ml----]
-plate 1 concentrations backwards -plate 3 had 2x exposure of diff concentrations, not run on flow
7/30/14
Flow Cytometry of PTEF1 + rtTA + pTET + GFP CB008 -could not run b/c cultures did not grow -original patch plates did not have much growth at all -restreaked plates
8/1/14
Cloning AFRP + Ste2 -doing this to increase receptor levels to alpha factor
Materials: 1X
5x HF Phusion Buffer 10 µl
10mM dNTPs 1 µl
10mM FW Primer 2.5 µl
10mM REV Primer 2.5 µl
Phusion Polymerase 0.5 µl
Template DNA 0.5 µl
ddH20 33 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Gel:
8/4/14
Flow Cytometry of Consti+rtTA+pTET+GFP CB008 Dox run
Plate 1:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008]--------------------]
B [---------------[CB008 pTET_GFP]-----------]
C [---------------[CB008 pTET_GFP pTEF1]-----]
D [---------------[CB008 pTET_GFP pTEF1 m6]--]
E [---------------[CB008 pTET_GFP pTEF1 m7]--]
F [---------------[CB008 pTET_GFP pTEF1 m10]-]
G
H
Plate 2:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008]--------------------]
B [---------------[CB008 pTET_GFP]-----------]
C [---------------[CB008 pTET_GFP pTEF1]-----]
D [---------------[CB008 pTET_GFP pTEF1 m6]--]
E [---------------[CB008 pTET_GFP pTEF1 m7]--]
F [---------------[CB008 pTET_GFP pTEF1 m10]-]
G
H
Plate 3:
1 2 3 4 5 6 7 8 9 10 11 12
A [---------------[CB008]--------------------]
B [---------------[CB008 pTET_GFP]-----------]
C [---------------[CB008 pTET_GFP pTEF1]-----]
D [---------------[CB008 pTET_GFP pTEF1 m6]--]
E [---------------[CB008 pTET_GFP pTEF1 m7]--]
F [---------------[CB008 pTET_GFP pTEF1 m10]-]
G
H
Doxycycline concentrations plate map:
H G F E D C B A
1 [----0 µg/ml----]
2 [---0.03 µg/ml--]
3 [---0.06 µg/ml--]
4 [---0.09 µg/ml--]
5 [---0.3 µg/ml---]
6 [---0.6 µg/ml---]
7 [---0.9 µg/ml---]
8 [---3.0 µg/ml---]
9 [---6.0 µg/ml---]
10 [---9.0 µg/ml---]
11 [---30 µg/ml----]
12 [---60 µg/ml----]
8/6/14
Colony PCR of AFRP + Ste2 Materials: 1X
GoTAQ Mix 10 µl
10mM FW Primer 1 µl
10mM REV Primer 1 µl
Template DNA 3 µl
ddH20 5 µl
PCR Cycle:
98°C 30 sec
98°C 10 sec
55°C 20 sec
72°C 30 sec
72°C 5 min
4°C ∞
Gel:
8/8/14
CB008 pTEF1+rtTA pTET+GFP pTET+MFalpha pAGA1+mCherry
Time points with [Dox] induction: 0, 1.5hr, 3hr, 5hr
Plate map for all time points (4 plates):
1 2 3 4 5 6 7 8 9 10 11 12
A [CB008 m6-1]----] [CB008 m10-3]----]
B [CB008 m6-2]----] [CB008DB m7-1]---]
C [CB008 m6-3]----] [CB008DB m7-2]---]
D [CB008 m7-1]----] [CB008DB m7-3]---]
E [CB008 m7-2]----]
F [CB008 m7-3]----]
G [CB008 m10-1]---]
H [CB008 m10-2]---]
[Dox] Concentration Map:
H G F E D C B A
1 [--------0 µg/ml------------]
2 [--------0.03 µg/ml---------]
3 [--------0.06 µg/ml---------]
4 [--------0.09 µg/ml---------]
5 [--------0.6 µg/ml----------]
6
7 [--------0 µg/ml------------]
8 [--------0.03 µg/ml---------]
9 [--------0.06 µg/ml---------]
10 [--------0.09 µg/ml---------]
11 [--------0.6 µg/ml----------]
12
-diluted 1:200 to get OD of 0.1 -fix with cycloheximide at diff. time points
8/11/14
Flow Cytometry of AFRP + rtTA + pTET + GFP -flow of 4 AFRPs: pHYM1, pYDR124W, pCLG1, pASG7 -plates are the same for all 4 promoters -1:200 dilution
Plate Map:
H G F E D C B A
1 [----0 µg/ml------------]
2 [----0.03 µg/ml---------]
3 [----0.06 µg/ml---------]
4 [----0.09 µg/ml---------]
5 [----0.3 µg/ml----------]
6 [----0.6 µg/ml----------]
7 [----0.9 µg/ml----------]
8 [----3.0 µg/ml----------]
9 [----6.0 µg/ml----------]
10 [----9.0 µg/ml----------]
11 [----30 µg/ml-----------]
12 [----60 µg/ml-----------]
Alpha Factor Concentration Map
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
8/12/14
Flow Cytometry of AFRP + rtTA + pTET + GFP -flow of 3 AFRPs: PRM1, PRM2, PRM3 -plates are the same for all 3 promoters
Plate Map:
H G F E D C B A
1 [----0 µg/ml------------]
2 [----0.03 µg/ml---------]
3 [----0.06 µg/ml---------]
4 [----0.09 µg/ml---------]
5 [----0.3 µg/ml----------]
6 [----0.6 µg/ml----------]
7 [----0.9 µg/ml----------]
8 [----3.0 µg/ml----------]
9 [----6.0 µg/ml----------]
10 [----9.0 µg/ml----------]
11 [----30 µg/ml-----------]
12 [----60 µg/ml-----------]
Alpha Factor Concentration Map
1 2 3 4 5 6 7 8 9 10 11 12
A [-------------------{0 nM}-------------------]
B [-------------------{0.5 nM}-----------------]
C [-------------------{1 nM}-------------------]
D [-------------------{10 nM}------------------]
E [-------------------{100 nM}-----------------]
F [-------------------{1000 nM}----------------]
G [-------------------{3000 nM}----------------]
H
8/13/14
Flow Cytometry of pTEF1 + rtTA pTET + GFP pTET + MFalpha pAGA1_mCherry CB008DB -1:200 dilution -Time points for Dox induction: 0, 1.5hr, 3hr, 5hr -plate map same for all 4 plates
Plate Map:
1 2 3 4 5 6 7 8 9 10 11 12
A [CB008 pTEF1-1]-] [CB008DB m3-3]--]
B [CB008 pTEF1-2]-] [CB008DB m6-1]---]
C [CB008 pTEF1-3]-] [CB008DB m6-2]---]
D [CB008DB pTEF1-1] [CB008DB m6-3]---]
E [CB008DB pTEF1-2]
F [CB008DB pTEF1-3]
G [CB008DB m3-1]--]
H [CB008DB m3-2]--]
Dox Concentrations:
H G F E D C B A
1 [--------0 µg/ml------------]
2 [--------0.03 µg/ml---------]
3 [--------0.06 µg/ml---------]
4 [--------0.09 µg/ml---------]
5 [--------0.6 µg/ml----------]
6
7 [--------0 µg/ml------------]
8 [--------0.03 µg/ml---------]
9 [--------0.06 µg/ml---------]
10 [--------0.09 µg/ml---------]
11 [--------0.6 µg/ml----------]
12
-0 time point plate was not fully run. Due to mistakes, only 10µl run on flow. plates saved to run flow on 0 time point plate tomorrow
8/14/14
Flow Cytometry of pTEF1 + rtTA pTET + GFP pTET + MFalpha pAGA1_mCherry CB008 -1:200 dilution -Time points for Dox induction: 0, 1.5hr, 3hr, 5hr -plate map same for all 4 plates -in 2ml culture plate there was a lot of debris/contamination in rows A,B,C -tried to avoid it by pipeting one at a time
Plate Map
1 2 3 4 5 6 7 8 9 10 11 12
A [CB008 pTEF1-1]-] [CB008 m7-3]-----]
B [CB008 pTEF1-2]-] [CB008 m10-1]----]
C [CB008 pTEF1-3]-] [CB008 m10-2]----]
D [CB008 m6-1]----] [CB008 m10-3]----]
E [CB008 m6-2]----]
F [CB008 m6-3]----]
G [CB008 m7-1]----]
H [CB008 m7-2]----]
Dox Concentrations:
H G F E D C B A
1 [--------0 µg/ml------------]
2 [--------0.03 µg/ml---------]
3 [--------0.06 µg/ml---------]
4 [--------0.09 µg/ml---------]
5 [--------0.6 µg/ml----------]
6
7 [--------0 µg/ml------------]
8 [--------0.03 µg/ml---------]
9 [--------0.06 µg/ml---------]
10 [--------0.09 µg/ml---------]
11 [--------0.6 µg/ml----------]
12
-flow experiment completely failed due to high bacterial contamination
8/15/14
Official Last Day of iGEM
