Team:UCSF UCB/eleanor

From 2014.igem.org

Eleanor's Lab Notebook

June 9, 2014

PCR for Constitutive Promoters (pTEF1(M6))

Materials 1x reaction 4.5x Master Mix
5x Phusion HF Buffer 10 µl 45 µl
dNTP's (10 mM) 1 µl 4.5 µl
Forward Primer (10µm) 2.5 µl 11.25 µl
Reverse Primer (10µm) 2.5 µl 11.25 µl
*Template DNA 0.3 µl 1.35 µl
Phusion Polymerase 0.5 µl 2.25 µl
Water 33.2 µl 149.4 µl
Total 50 µl 225 µl

*Added everything to template DNA. oops

  1. Mix materials in a 4.5x Master Mix on ice. Mix well.
  2. Pipetter 50 µl from the Master Mix into 4 labeled PCR tubes

  3. Thermocycler for : 

    Initial Duration   | 98° C | 30s 
  35 Cycles of: 
      Denaturation | 98° C | 10s
      Annealing    | 55° C | 20s
      Extension    | 72° C | 30s
Final Extension    | 72° C | 5m
Hold               | 4°  C | Forever

  4. Keep Samples for gel extraction on tje next day

June 10, 2014

Gel of Constitutive Promoters

  1. Add 5 µl Bluejuice 10x to each of the tubes
  2. Load all on gel.

Constitutive Promoters Gel

Gel Extraction

QlAquick Gel Extraction Kit

  1. Cut Gel
  2. Weigh it in a colorless tube
  3. Add 3 volumes Buffer Q G to 1 volime Gel (100mg ~ 100µl)
  4. Incubate @ 50° C for 10 min or until completely dissolved (vortex every 2-3 min to help dissolve)
  5. Add 1 gel volume isopropanal to the sample and mix
  6. Place a QlAquick soin column in a provided 2ml collection tube
  7. Place sample in column & spin for 1 min --> discard flow through
  8. To wash add 0.75 ml Buffer PE to column & centrifue for 1 min, then dry spin
  9. Place column in 1.5 ml tube
  10. Add 35µl H2O & centrifuge for 1 min.

Restriction Enzyme Digest with APA1

  • 40 µl DNA
  • 5 µl Cutsmart
  • 0.5 µl APA1 Room Teperature Overnight

June 11, 2014

Digest with Xho1

-0.5µl let sit for a few hours

PCR Purification

  1. 5 volumes of Buffer PB (with ethanol) to 1 volume PCR sample
  2. Apply sample to column and spin for 1 min
  3. Discard flow through
  4. To wash add 0.75 ml of Buffer PE to column and centrifuge for 1 min
  5. Discard flow through and place column back in the same tube
  6. Cetrifuge for an addtitional minute (Dry Spin)
  7. Place column in clean 1.5 ml tube
  8. [Elute DNA] Add 30µl water to column, let stand for 1 min and centrifuge for 1 min

M6 + PSV606 ligation

1:3 backbone + insert

insert concentration: 138 ng/µl

Reagents 1x
10x ligase buffer 1.0µl
DNA Backbone (PSV606) 0.2µl
Insert 0.2µl
T4 DNA Ligase 0.5µl
H2O 8.1µl
Total 10 µl

Room Temperature for 2 hours

E. Coli Transformation

 - 10 µl ligation
 - 50 µl competent cells
     --> 30m on ice
     --> 45s heatshock 42° C
     --> 2m on ice
 - 250 µl of SOC media
     --> 1h shake 37° C
 Plate

June 12, 2014

  • Colonies didn't grow
    • Possibly because Kara gave us cheap cells to use
  • Redo:
    • ligation - 0.4 µl backbone & 7.9 µl H2O
    • transformation - used more expensive competent cells

June 13, 2014

Yeast Transformation (CB008 + Inducible Promoters)

Reagents
YPD
1 M LiOAc
10X TE pH 7.5
1X TE pH 7.5, 0.1 M LiOAc
50% PEG 3350
DMSO
Salmon Sperm DNA (ssDNA)
  • PEG = viscous, pipette slow
  • boil ssDNA aliquots

Previous Day : Grow yeast strain to be transformed in 5-10 mL YPD overnight at 30° C

  1. Set up digest to linearize DNA
  2. Dilute O/N culture ~ 1:20 in YPD grow 2-4 hours at 30° C
  3. Prepare ssDNA - boil for 10m cool on ice for at least 10m (10 µl of 10 mg/mL stock per transformation)
  4. Harvest cells in centrifuge - 3000 rpm, 2-5 min
  5. Wash with 1 ml 0.1 M LiOAin TE
  6. Pellet cells - 3000 rpm , 2-5 min
  7. Resuspend pellet in 100 µl 0.1 M LiOAc in TE per 2.5 ml culture, split into 100 µl per epindorph tube for each transformation
  8. to 100 µl cells add 100 µg ssDNA (10 µl of 10 mg/mL sock), 1-5 µl target DNA
  9. Add (in order): 480 µl 50% PEG 3350, 60 µl 10X TE, 60 µl 1 M LiOAc (for final 40% PEG, 1X TE, 0.1 M LiOAc) Optional: Add 75 µl DMSO definitely did
  10. Vortex
  11. Incubate 42° C for 30m & begin drying plates
  12. Pellet (6000 rpm - 2m), discard supernatant (remove PEG completely by pipetting), resuspend in 500 µl YPD (or selective media)
  13. Pellet, discard supernatant, resuspend in risidual ~ 50 µl YPD
  14. Plate on selective media
  15. Incubate 1-3 days

June 16, 2014

Colony PCR for screening E. Coli (Constitutive promoters + GFP)

Pick single colonies ( 5 from each plate ) mix in 25 µl H2O in a tube. Use 5 µl in PCR reaction

Reagents 1X 6X
2X GoTaq Green PCR Master Mix 10 µl 60 µl
10 µM Forward primer 1 µl 6 µl
10 µM Reverse primer 1 µl 6 µl
Water 3 µl 18 µl
Bacterial cells (template) 5 µl -----

Cycle: 

95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever

load 5 µl onto gel

for all positive bands - take the rest of the cells from step 1 and inoculate them into an overnight LB (+antibiotic) for miniprep

Colony PCR for E. Coli 6/16/14

Colony PCR for Yeast & Patching

  1. Number colonies
  2. Patch on plate
  3. Mix in 10 µl NaOH
  4. Boil for 20m
  5. PCR

Cycle: 

95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever

Only 9 lanes worked Gel 1: SAG(1), ~~PRM3(1)~~, ASG7(1), ~~PRM3(3)~~, PCL2(2) [PRM3 mislabeled possibility of one being PRM6. unusable. Gel 2: EXM18(1), ECM18(2), PRM2(3), CLG(3) will redo

June 17, 2014

CB008(x) CB008DB(DB)
CLG1 PRM1
PRM1 YDR124W
PRM3 PRM6
PRM2 HYM1
ECM18 PCL2
YDR124W SAG1
PRM6 PRM3
-------- ASG7
  • Create new template by picking off patch plate
  • also pick 2 new colonies (4&5) from original growth plate and create new patch plate

Gel Photos

Colony PCR for Yeast Inducible Promoters 6/17/14

Colony PCR for Yeast Inducible Promoters 6/17/14

Colony PCR for Yeast Inducible Promoters 6/17/14

June 18, 2014

Frozen Glycerol Stocks Yeast

1) Grow Overnight in YPD (2-5 mL) then dilute 1:20 in YPD, grow to OD 0.4-0.5 2) Add 350 µl cells to 350 µl sterile 60% glycerol in cryovial, vortex to mix, snap freeze in liquid nitrogen and store at -80° C (but actually we just stuck it in the freezer because nobody will let us use liquid nitrogen).

Constitutive Promoters Miniprep

  1. Resuspend pelleted bacterial cells in 250 µl Buffer PI and transfer to a microcentrifuge tube.
  2. Add 250 µl Buffer P2 and gently invert tube 4-6 times to mix
  3. Add 350 µl Buffer N3 and invert immediately 4-6 times
  4. Centrifuge for 10 min at 13,000 rpm
  5. Add supernatant to QIAprep spin column
  6. Centrifuge for 30-60s - Discard flow through
  7. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s - Discard flow through
  8. Wash column by adding 0.75 ml Buffer PE and centrifuginh for 30-60s
  9. Discard flow through and centrifuge for an additional 1 min to remove residual wash buffer
  10. Place column in clean 1.5 ml microcentrifuge tube. To elute DNA add 50 µl water or buffer EB, let stand for 1 min and centrifuge for 1 min.

June 19, 2014

Adding α Factor to CB008 Promoters and Flow Cytometry

Concentrations of α factor 3mM stock 0, 1 nm, 10 nm, 100 nm, 1 µm We want to use 10 µl of 100x concentration 100nm, 1000nm, 10 µm, 100 µm

Amount of reagents desired concentration
30µl of 3mM + 870 µl H2O 100 uM
50 µl of 100 µm + 450 H2O 10 µM
50 µl of 10 µm + 450 H20 1000 nM
50 µl of 1000 nm + 450 H2O 100nM 
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-----]

Protocol : Night Before - Start overnight culture in SD coplete media (not YPD)

Day of: 1. Dilute cultures to final ~OD 0.05 - 0.1 in the 96 well shaker plate (saturated culture) should be ~OD 7 2. Allow cells to enter growth stage by putting on plate shaker for 3 hrs at 100 rpm 30° C 3. Induce w/ α factor concentrations [0 , 1nm, 10nm, 100nm, 1µm] 4. Allow induction to proceed on shaker for 90 min to 2 hrs 5. Transfer 250 µl of each culture to 96 well flow cytometry V-bottom plate using a multichannel pipette. Also arrested cells w/ cyclohexiimide - 10ml/well 6. Run on Flow Cytometer - Check fluid levels, press run - check voltage 7. Analyze data -> FloJo/MatLab

Flow Voltages Inspector : FSC: 250 SSC: 280 FITC: 550

Everything

yGEM16

June 20, 2014

Yeast Transformation w/ Constitutive Promoters

PTEF1, M3, M6, M7, M10 in CB008 and CB008DB

  1. Linearize Plasmids - PSV606 10 µl DNA 5 µl Cutsmart Buffer 1 µl PME1 34 µl H2O 37 ° C for 2 hours

Procedure the same as June 13, 2014

June 23, 2014

Colony PCR and Gels (Constitutive Yeast)

  • protocol same as June 16

Gel Picture

  • Overnight Cultures in 5 ml YPD

June 25, 2014

Glycerol Stocks of Constitutive Promoters in Yeast

  • dilute cultures ~ 1:20 4-5 hours before
  • 420 µl of 50% glycerol + 350 µl cells
  • vortex
  • store in -80° C freezer

m3 leaked during dilution

Parts Registry Inducible Promoters PCR with pSB1C3 overhang

Reagents 1x 12x
5x phusion HF Buffer 10 µl 120 µl
dNTP's (10 mM) 1 µl 12 µl
Phusion Polymerase 0.5 µl 6 µl
Water 33.2 µl 398.4 
  .3 µl template DNA 
  44.7 µl master mix
  2.5 µl FW Primer
  2.5 µl RV Primer


    Initial Denaturation  | 98° C | 30s 
      35 Cycles of: 
          Denaturation    | 98° C | 10s
          Annealing       | 55° C | 20s
          Extension       | 72° C | 30s
    Final Extension       | 72° C | 5m
    Hold                  | 4°  C | Forever

25µl sybersafe used when pouring gel

Gel Picture

«Only pGEM's 2 & 3 worked»

June 26, 2014

PCR Constitutive and Inducible Promoters with PSB1C3 overhang

« pGEM's 1,4,5,6,7,8,9,10,11,12,13,14,15,16 »

Reagents 1x 17X
Template DNA 0.3 µl N/A
FW Primer 2.5 µl N/A
RV Primer 2.5 µl N/A
DMSO 1.5 µl 25.5 µl
5x Phusion HF Buffer 10 µl 170 µl
dNTP's 1 µl 17 µl
Phusion Polymerase 0.5 µl 8.5 µl
Water 31.7 µl 538.9 µl 
   Initial Denaturation   | 98° C | 30s 
      35 Cycles of: 
          Denaturation    | 98° C | 10s
          Annealing       | 55° C | 20s
          Extension       | 72° C | 30s
    Final Extension       | 72° C | 5m
    Hold                  | 4°  C | Forever

June 27, 2014

Gel Picture

«Still need to redo pGEM 5,6,8»

Constitutive Promoter Digest

pGEM12 - pGEM16

  • Removing GFP to insert rtTA

    Not1 HF & Xho1

    Reagents 1x
    DNA 15 µl
    Cutsmart 2.5 µl
    Xho1 0.5 µl
    Not1 HF 0.5 µl
    6.5 µl H20

    incubate at 37° C for ~ 2 hours

Parts Registry PCR Redo & PCR Purification

«Same protocol as on the 26th»

Gel Picture

#finally

PCR Purification (pGEM 1,2,3,4,7,9,10-16) ( Same as June 11)

  1. 5 volumes of Buffer PB (with ethanol) to 1 volume PCR sample
  2. Apply sample to column and spin for 1 min
  3. Discard flow through
  4. To wash add 0.75 ml of Buffer PE to column and centrifuge for 1 min
  5. Discard flow through and place column back in the same tube
  6. Cetrifuge for an addtitional minute (Dry Spin)
  7. Place column in clean 1.5 ml tube
  8. [Elute DNA] Add 30µl water to column, let stand for 1 min and centrifuge for 1 min

Gibson Assembly

Reagents 1X
pSB1C3 (25ng/µl) 2 µl
insert 3 µl
Gibson Master Mix 5 µl

50° C fro 1 hour -> ice

Transformation

    10µl Gibson
    25 µl Mach I cells

    30 m ice
    45 s heat shock (42° C)

    2 m ice
    +250 SOC Media

    1 hr. 37°C

    Plate on LB + CAM 
    Keep in drawer over the weekend
  • Didn't Work

June 30, 2014

Colony PCR for Yeast ( pTET GFP & pAGA GFP CB008 + CB008 DB )

  1. Number colonies
  2. Patch on plate
  3. Mix in 10 µl NaOH
  4. Boil for 20m
  5. PCR

Cycle: 

95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever

pTET patch plate -leu had an orange and tellow stripe which was kind of sketchy. It isnt growing either so we are going to replate from one of the original colonies just to check. (7/2/14)

Primers Used - pAGA - regular pSV606 primers pTET - pTET primers

July 1, 2014

pTET-GFP/pAGA-GFP Colony PCR's

Gel Picture

Making iGEM Backbone

5 tubes - 50 µl reaction each

Reagents 1x 5x
Phusion Polymerase 0.5 µl 2.5 µl
Template DNA 1 µl 5 µl
Phusion HF Buffer 10 µl 50 µl
dNTP's 1 µl 5 µl
FW Primer (SB prep 3P1) 2.5 µl 12.5 µl
RV Primer (SB Prep 2-Ga) 2.5 µl 12.5 µl
Water 32.5 µl 162.5 µl

*iGEM provides different template DNA than pSB1C3

Initial Denaturation   | 98° C | 30s 
      35 Cycles of: 
          Denaturation    | 98° C | 10s
          Annealing       | 55° C | 20s
          Extension       | 72° C | 30s
    Final Extension       | 72° C | 5m
    Hold                  | 4°  C | Forever

Different times for a bulk reaction but we went with the one above

-Failed

  • Redone 7/2/14 using pSB1C3 as template 7/2/14

July 2, 2014

Glycerol Stocks for pTET-GFP & pAGA-GFP

«still need to check if pTET is okay (patch plate)»

PCR Cleanup of pSB1C3

  1. 5 volumes of Buffer PB (with ethanol) to 1 volume PCR sample
  2. Apply sample to column and spin for 1 min
  3. Discard flow through
  4. To wash add 0.75 ml of Buffer PE to column and centrifuge for 1 min
  5. Discard flow through and place column back in the same tube
  6. Cetrifuge for an addtitional minute (Dry Spin)
  7. Place column in clean 1.5 ml tube
  8. [Elute DNA] Add 30µl water to column, let stand for 1 min and centrifuge for 1 min

Digestion of pGEM insert with pSB1C3 overhang and pSB1C3 backbone

Gibson didn't work, so we are going the standard "cut & paste" route

Reaction is as follows:

            5 µl cutsmart
            1 µl enzymes
            _ µl H20
            _ µl DNA
        + ___________
           50 µl Total 

 We want 1000 ng of DNA
pGEM # concentration µl for digestion µl H20
1 245.1 ng/µl 4.1 µl 39.9 µl
2 116.9 ng/µl 8.6 µl 35.4 µl
3 81.77 ng/µl 12.3 µl 31.7 µl
4 87.00 ng/µl 11.5 µl 32.5 µl
5 12.26 ng/µl 81µl* (30µl) 14 µl
6 60.79 ng/µl 16.45 µl 27.5 µl
8 55.62 ng/µl 18 µl 26 µl
9 87.86 ng/µl 11.38 µl 32.62 µl
10 25.02 ng/µl 40µl* (30µl) 14 µl
11 54.95 ng/µl 18.2 µl 25.8 µl
12 45.29 ng/µl 22.1 µl 21.9 µl
13 55.37 ng/µl 18.06 µl 25.94 µl
14 35.62 ng/µl 28.07 µl 15.93 µl
15 107.1 ng/µl 9.3 µl 34.7 µl
16 24.08 ng/µl 41.52µl* (30 µl) 14 µl

37° C for 2 hours or overnight

Wanted to keep DNA 30 µl or under*

«pGEM7 wasn't used because it isnt biobrick compatible»

July 3 - July 6, 2014

-out- Check Roberts Lab Notebook for what happened

(He ligated and transformed)

July 7, 2014

All plates grew including the negative control.

Colony PCR

Pick single colonies ( 5 or so from each plate ) mix in 25 µl H2O in a tube. Use 5 µl in PCR reaction

Reagents 1X 6X
2X GoTaq Green PCR Master Mix 10 µl 60 µl
10 µM Forward primer 1 µl 6 µl
10 µM Reverse primer 1 µl 6 µl
Water 3 µl 18 µl
Bacterial cells (template) 5 µl -----

Cycle: 

95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever

Gel Picture

Gel Picture

Made liquid cultures of ones that worked Re - Colony PCR pGEM 3,10,13

July 8, 2014

Liquid cultures didn't work

  • Used LB-Carb instead of LB Cam -oops- Re-Culturing

Colony PCR (Redo pGEM's 3, 10, 13)

Gel Picture

  • Made cultures of 3 & 10
  • Redo 13 (13.7-13.11 labelled 1-5)

Gel Picture

July 9, 2014

Parts Registry Miniprep

pGEM # Colony #
1 1
2 1
3 4
4 1
5 1
6 1
7 1
8 1
9 1
10 4
11 1
12 1
14 1
15 1
16 1
  1. Resuspend pelleted bacterial cells in 250 µl Buffer PI and transfer to a microcentrifuge tube.
  2. Add 250 µl Buffer P2 and gently invert tube 4-6 times to mix
  3. Add 350 µl Buffer N3 and invert immediately 4-6 times
  4. Centrifuge for 10 min at 13,000 rpm
  5. Add supernatant to QIAprep spin column
  6. Centrifuge for 30-60s - Discard flow through
  7. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s - Discard flow through
  8. Wash column by adding 0.75 ml Buffer PE and centrifuginh for 30-60s
  9. Discard flow through and centrifuge for an additional 1 min to remove residual wash buffer
  10. Place column in clean 1.5 ml microcentrifuge tube. To elute DNA add 50 µl water or buffer EB, let stand for 1 min and centrifuge for 1 min.

Transformation of pGEM 13

10µl plasmid
25 µl cells

30m on ice
45s heat shock
2m on ice

+250 µl SOC Media

1hr. 37° C
Plate on LB CAM

July 10, 2014

Colony PCR and overnight cultures

Gel Picture

( shared gel with Jessica )

July 11, 2014

Miniprep pGEM 13

  1. Resuspend pelleted bacterial cells in 250 µl Buffer PI and transfer to a microcentrifuge tube.
  2. Add 250 µl Buffer P2 and gently invert tube 4-6 times to mix
  3. Add 350 µl Buffer N3 and invert immediately 4-6 times
  4. Centrifuge for 10 min at 13,000 rpm
  5. Add supernatant to QIAprep spin column
  6. Centrifuge for 30-60s - Discard flow through
  7. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s - Discard flow through
  8. Wash column by adding 0.75 ml Buffer PE and centrifuginh for 30-60s
  9. Discard flow through and centrifuge for an additional 1 min to remove residual wash buffer
  10. Place column in clean 1.5 ml microcentrifuge tube. To elute DNA add 50 µl water or buffer EB, let stand for 1 min and centrifuge for 1 min.

July 12, 2014

Check Sequencing and nanodrop of all parts(concentrations written on tubes)

July 17, 2014

500µl Carb + 500ml LB = LB + Carb

July 21, 2014

Colony PCR PTET-GFP + pTET-mfα + Constitutive Promoter-rtTA

CB008 CB008DB
PTEF1 m6
m6 m7
m7 -
m10 -

others are still being made

only 3 worked - m7 #2 & m10 #3 & #4

Gel Picture

Will repeat

July 22, 2o14

Gel Picture

July 23, 2014

Fluorescent Protein Transformations for Exploratoreum

  • GFP
  • Citrine
  • mT BFP2
  • mRuby
  • ECFP
  • mKitr

  • EGFP 

     .5 µl DNA
 25 µl cells

 30m on ice
 45s heat shock
 2m on ice

+250 µl SOC Media

1hr. 37° C
Plate

    Didn't work. Found in old UCSF iGEM teams stuff. Possibly for yeast.

July 25, 2014

Yeast Transformations

pTET-mfα in 11E3128 into pAGA1-mCherry into
pTEF1 DB pTEF1
pTEF1(m10) DB pTEF1(m6)
- pTEF1(m7)
- pTEF1(m10)
- pTEF1(m6) DB
- pTEF1(m7) DB

Digest w/ PME1

5µl 11E3128 
1µl PME1
1µl cutsmart
3µl H20

and 

10µl pTET-GFP pTET mfα
1 µl PME1
1.5µl cutsmart
2.5µl H20

37° C for 1h

E. Coli Transformation pGEM 22 ( pAGA1-mCherry)

0.2 µl plasmid
25 µl cells (DH5α)

30m ice
45s 42°C
2m ice

+250 µl SOC Media
1h 37°C

Plate on LB Carb
Place in drawer over the weekend

August 5, 2014

Streak Plates for Jessica

CB008 pTET+GFP AFRP +rtTA
PRM1
PRM2
PRM3
YDR124W
CLG1
ASG7
HYM1

Glycerol Stocks of

  • pTET+GFP + m3+rtTA + pTET+mfAlpha
  • pTET+GFP + m10+rtTA + pTET+mfAlpha + pAGA mCherry

Streak & Overnight culture of m6 & m7 CB008 & DB pTET GFP +rtTA +mfalpha + mCherry

August 6, 2014

Glycerol Stocks of

  • CB008 m6/m7+(GFP rtTA mfalpha)+pAGA mCherry
  • CB008DB m7+(GFP rtTA mfalpha)+pAGA mCherry

August 8, 2014

BFP Miniprep

August 11, 2014

Flow Cytometry Set Up

pTET GFP

Dox and alpha factor inductions

YDR124W, pASG7, HYM1, CLG1

Dox 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 waterb
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          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

Alpha Factor Dilutions :

1x 100x
0 nm 0 nm
0.5 nm 50 nm
1 nm 100 nm
10 nm 1000 nm
100 nm 10000 nm
1000 nm 100000 nm
3000 nm 300000 nm

30 µl 3Mm stock + 270 µl H2O = 300,000 nm

100 µl 300,000 nm stock + 200 µl H2O = 100,000 nm

50 µl 100,000 nm + 450 µl H2O = 10,000 nm

50 µl 10,000 nm + 450 µl H2O = 1,000 nm

50 µl 1000 nm + 450 µl H2O = 100 nm

200 µl 100 nm + 200 µl H20 + 50 nm

Cutures of AFRP's + rtTA & pTET GFP (PRM1, PRM2 , PRM3)

August 13, 2014

Flow

Const. +rtTA mCherry pTET GFP

In triplicate

Time Course

0, 1.5, 3, 5 hours

dox concentrations

0, 0.03, 0.06, 0.09, .6, 6

pTEF1, m3, m6, m7, m10

August 14, 2014

Flow

Const. + mCherry

Same as August 13 except includes 8hr time point.

Weird clumps found in shaker plate - cancelled

August 15, 2014

1:200 dilutions 7:50 AM

Redo of August 14

  1  2  3  4  5  6  7  8  9  10 11 12 
A { --- pTEF 1 ---} { ----- m7 ------}
B { --- pTEF 1 ---} { ----- m10 -----}
C { --- pTEF 1 ---} { ----- m10 -----}
D { ----- m6 -----} { ----- m10 -----}
E { ----- m6 -----}
F { ----- m6 -----}
G { ----- m7 -----}
H { ----- m7 -----}

 dox concentration increases from left to right. 
 0, 0.03, 0.06, 0.09, 0.6, 6

August 19, 2014

Flow

1:200 dilution 7:40 AM

mCherry( +pTET GFP mfAlpha const. rtTA) DB strains

 1  2  3  4  5  6  7  8  9  10 11 12 
A { --- pTEF 1 ---} { ----- m6 ------}
B { --- pTEF 1 ---} { ----- m7 ------}
C { --- pTEF 1 ---} { ----- m7 ------}
D { ----- m3 -----} { ----- m7 ------}
E { ----- m3 -----} { ----- m10 -----}
F { ----- m3 -----} { ----- m10 -----}
G { ----- m6 -----} { ----- m10 -----}
H { ----- m6 -----}

 dox concentration increases from left to right. 
 0, 0.03, 0.06, 0.09, 0.6, 6

 0, 1.5, 3, 5, 8 hours

August 28, 2014

Start cultures for CB008 const. +rtTA pTET GFP pTET mfAlpha for PCL2 mCherry transformation

Flow data does not show RFP - same for m6 pAGA mCherry

  • streak yeast strains from glycerol stocks - yGEM's 17, 20, 53, 54, 68, 69, 70, 88, 96

August 29, 2014

Transform yGEM 47-50 + PCL2 mCherry Const. rtTA, pTET GFP, pTET mfAlpha

September 2, 2014

  • Forgot to linearize plasmid August 29 - will colony PCR since some colonies grew but probably didn't work

  • Prepared dilutions to retransform using linearized plasmid in Jeffrey's box

September 4, 2014

  • Colony PCR PCL2 mCherry's

    Transform :

    CB008 m3 rtTA w/ pTET mfAlpha

    CB008 m6 mfAlpha w/ pAGA1 mCherry

    linearized plasmid in Jeffrey's box

    mfAlpha auxotrophic marker - HIS

    mCherry on TRP site

    Gel Photo machine wasn't working - pTEF (1,2,3) and m7 (3) worked

    Redo m6 & m10

    Overnight cultures od pTEF&m7&m10 pAGA mCherry for flow

September 5, 2014

Flow (Co-cultures) pAGA mCherry CBoo8

  • Same Dox concentrations
  • 1.5 ml SD in the growth plate

  • final dilution OD 0.03 so OD 0.015 for each strain 

      1  2  3  4  5  6  7  8  9 10 11 12
A { -pTEF1 x M7- }  { -pTEF1 x M10- }
B { -pTEF1 x M7- }  { -pTEF1 x M10- }
C { -pTEF1 x M7- }  { -pTEF1 x M10- }
D { --M7 x M10-- }
E { --M7 x M10-- }
F { --M7 x M10-- }
G
H
x ptef1 m7 m10
OD 0.39 0.39 0.39
Actual OD 3.9 3.9 3.9

Actual OD / Target OD = Dilution Factor

3.9/0.015 = 260

µl / Dilution Factor = µl to be added

1500 µl/260 = 5.77 µl

Colony PCR

  • Failed

  • Try again with Zymalase

     25 µl Zymalase
 + Cells

 37 °C 10m

 5 µl ^ in PCR reaction 

 Only m10 b/c m6 is growing weird

Need to:

  • Retransform M6 with PCL2 mCherry
  • Colony PCR m3 mfAlpha & m6 pAGA RFP

All m6-mfAlpha strains when transformed grow weird

September 8, 2014

Streak Plates of yGEM 75 & 76

Colony PCR - m3 w/ mfAlpha retransform m6 w/ pAGA1/pPCL2 +mCherry

-Grows weird

Transform m3+mfAlpha

September 9, 2014

Flow - New PCL2s -m7 m10 pTEF1

September 10, 2014

yGEM 48 (m6-mfAlpha) developed a weird mutation allowing it to grow strangely on an SD-TRP plate without integrating the plasmid.

Will retransform m6-rtTA with mfAlpha

3 Transformations:

  • m6+rtTA w/mfAlpha
  • m6+rtTA w/mfAlpha & pCL2 mCherry
  • - m6+rtTA w/mfAlpha & pAGA1 mCherry

September 11, 2014

Flow and Transformations

      1  2  3  4  5  6  7  8  9  10 11 12 
A { --- pTEF 1 ---} { ----- m10 -----}
B { --- pTEF 1 ---} { --- m7+pTEF1 --}
C { --- pTEF 1 ---} { --- m7+pTEF1 --}
D { ----- m7 -----} { --- m7+pTEF1 --}
E { ----- m7 -----} { --- m10 + M7---}
F { ----- m7 -----} { --- m10 + M7---}
G { ----- m10 ----} { --- m10 + M7---}
H { ----- m10 ----}

dox concentration increases from left to right. 0, 0.03, 0.06, 0.09, 0.6, 6

0, 1.5, 3, 5, 8 hours Cancelled

September 14, 2014

Colony PCR with Zymalase

Reagents 1X 10X
2X GoTaq Green PCR Master Mix 10 µl 100µl
10 µM Forward primer 1 µl 10 µl
10 µM Reverse primer 1 µl 10 µl
Water 3 µl 30 µl
Bacterial cells (template) 5 µl ------
DMSO 1.5 µl 15 µl
Zymalase 0.5 µl 5 µl 
37° C | 5m
95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever
  • Didnt Work

September 15, 2014

Flow Repeat (9/11)

  1  2  3  4  5  6  7  8  9  10 11 12 
A { --- pTEF 1 ---} { ----- m10 -----}
B { --- pTEF 1 ---} { --- m7+pTEF1 --}
C { --- pTEF 1 ---} { --- m7+pTEF1 --}
D { ----- m7 -----} { --- m7+pTEF1 --}
E { ----- m7 -----} { --- m10 + M7---}
F { ----- m7 -----} { --- m10 + M7---}
G { ----- m10 ----} { --- m10 + M7---}
H { ----- m10 ----}
x ptef1 m7 m10
OD 0.64 0.67 0.67
Actual OD 6.4 6.7 6.7

Actual OD / Target OD = Dilution Factor

6.4/0.015 = 427
6.7/0.015 = 447

µl / Dilution Factor = µl to be added

1500/427 = 3.5µl
1500/447 = 3.4µl

Colony PCR from day before failed.

Repeat

To Check for mfAlpha

Reagents 1X 10X
2X GoTaq Green PCR Master Mix 10 µl 100 µl
10 µl RA146 1 µl 10 µl
10 µl RA145 1 µl 10 µl
Water 3 µl 30 µl
Bacterial cells (template) 5 µl ------

pAGA

Reagents 1X 6X
2X GoTaq Green PCR Master Mix 10 µl 50 µl
10 µl RA148 1 µl 5 µl
10 µl RA145 1 µl 5 µl
Water 3 µl 15 µl
Bacterial cells (template) 5 µl -----

pTET mfAlpha Digestion

Reagents 1X
plasmid 23 µl
Cutsmart 3 µl
PME1 1 µl
H2O 3 µl

September 22, 2014

Digest

PCL2 - Bar1 w/ APA1 & Not1

CLG1 - Bar1 w/ APA1 & Not1

CLG - Bar1 w/ Xho1 & Not 1

APA1 - 1hr RT Not 1 37° O/n

Not1 + Xho1 - 37° O/n

PJW608 APA1/Not1 HF

September 23, 2014

Gel Extraction

  • CLG - Bar1
  • PCL2 - Bar1
  • Bar1

PCR Purification

PJW608

Reagents Bar1 PCL2 CLG1
10x Buffer 2 µl 2 µl 2 µl
Vector DNA 1 µl 1 µl 1 µl
Insert DNA 6 µl 2.3 µl 1.6 µl
H20 10 µl 13.7 µl 14.4 µl
Ligase 1 µl 1 µl 1 µl

September 24, 2014

pAGA1 + Bar1 + PJW608

1 µl Ligase 2 µl Buffer 1 µl Vector 6.2 + x µl insert 9.8 µl H2O

Colony PCR

pCL2 Bar1 CLG Bar1

September 25, 2014

Colony PCR pAGA Bar1

LB Cultures: CLG Bar1 PCL2 Bar1

September 26, 2014

Miniprep PJW608 - CLG (or PCL2) Bar1

Transform into DB strains

  • linearize plasmids
    • 6 µl Plasmid
    • 2.5 µl cutsmart
    • 1 µl PME1
    • 13.5 µl H2O

Colony PCR pAGA Bar1 again

September 29, 2014

Colony PCR

CLG1 PCL2
m6 pTEF1
m3 m3
pTEF1 m6
----- m10

m10 CLG did not grow

September 30, 2014

Bar 1 Flow

  1  2  3  4  5  6  7  8  9  10 11 12 
A { --- pTEF 1 ---} { ----- m6 ------}
B { --- pTEF 1 ---} { ----- m10 -----}
C { --- pTEF 1 ---} { ----- m10 -----}
D { ----- m3 -----} { ----- m10 -----}
E { ----- m3 -----} { ---- m3+m6 ----}
F { ----- m3 -----} { ---- m3+m6 ----}
G { ----- m6 -----} { ---- m3+m6 ----}
H { ----- m6 -----}
x m3 m6
OD 0.4 0.45
Actual OD 4 4.5

Actual OD / Target OD = Dilution Factor

4/0.015 = 266.66
4.5/0.015 = 300

µl / Dilution Factor = µl to be added

1500/266.66 = 5.6 µl
1500/300 = 5 µl

October 2, 2014

Flow Cocultures w/ Dye

Stain m7 with blue dye (Cell Tracker?)

  • Find OD.
  • OD/0.015 = DF
  • 1000µl/DF = x per well
  • xµlperwell x 100 = y amount of overnight culture to dye
  • Split y into 2 1.5 ml tubes
  • Pellet - 8000 rpm for 3 min
  • Remove liquid, no cells
  • add 1 ml HEPES and 10 µl Cell Tracker to each tube
  • gently resuspend and keep at RT for 30m
  • Flick tube every 5m or so (protect from light)
  • Pellet
  • Remove Liquid and Replace w/ 1ml SD media
  • Pellet
  • Remove liquid
  • Add y/2 µl SD to each tube.

  • Resuspend 2xµl in m7 only wells (OD 0.03) xµl in coculture wells (OD 0.015)

       1  2  3  4  5  6  7  8  9  10 11 12 
A { --- pTEF 1 ---} { ----- m10 -----}
B { --- pTEF 1 ---} { --- m7+pTEF1 --}
C { --- pTEF 1 ---} { --- m7+pTEF1 --}
D { ----- m7 -----} { --- m7+pTEF1 --}
E { ----- m7 -----} { --- m10 + M7---}
F { ----- m7 -----} { --- m10 + M7---}
G { ----- m10 ----} { --- m10 + M7---}
H { ----- m10 ----}
x ptef1 m7 m10
OD 0.44 0.62 0.47
Actual OD 4.4 6.2 4.7

Actual OD / Target OD = Dilution Factor

4.4/0.015 = 293.33
6.2/0.015 = 413.33
4.7/0.015 = 313.33

µl / Dilution Factor = µl to be added

1000/293.33 = ~3.41µl
1000/413.33 = ~2.42µl
1000/313.33 = ~3.19µl

Retrieved from "http://2014.igem.org/Team:UCSF_UCB/eleanor"