Team:Cooper Union/Notebook/Telomere August

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<li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Biohack_project">Biohacker Kit </a></li>
<li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Biohack_project">Biohacker Kit </a></li>
<li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Hardware">OpenSource Hardware </a> </li>
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<li class="menu"> <a class="menu" href="https://2014.igem.org/Team:Cooper_Union/Parts">BioBrick Parts </a></li>
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<li class="menu-notebook">  Notebook  
<li class="menu-notebook">  Notebook  
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Revision as of 00:10, 18 October 2014

Cooper Union 2014 iGEM

Programmable Lifespan Timer

JUN JUL AUG SEP


8/1/14

  • Checked yeast cells in roller
  • counted cells with hemacytometer
    W303α: 1.31x108 cells/mL
    W303A: 1.4x108 cells/mL -- 5μL for dilution
    1296: 1.015x108 cells/mL -- 5μL for dilution
    1294: 2.805x106 cells/mL -- 214μL for dilution
    1297: 3.695x106 cells/mL -- Some of these yeast cells were dark and dying: this strain is in the middle of senescencing
  • Diluted W303A, 1296, 1294 in triplicate in 6mL new YPD media to cell density of 105

For tomorrow: count cells and re-dilute

8/2/2014


Cell count:
1296
1: 6.4x107
2: 1.63x108
3: 1.28x108
Ave: 1.18x108+0.15-0.18
W303A
1: 3.08x107
2: 3.72x107
3: 2.99x107
Ave: 3.26x107+0.15-0.09
1294
1: 1.72x108
2: 1.63x108
3:1.83x108
Ave: 1.73x108+0.03-0.03
Recultured with 3-1-1: closest to average
1296: 3.906μl
W303A: 16.26μl
1294: 29.07μl

8/3/2014


Cell count:
1296
1: 1.39x108
2: 1.39x108
3: 1.07x108
Ave: 1.28x108+0.04-0.07
W303A
1: 1.05x108
2: 9.2x107
3: 1.25x108
Ave: 1.07x108+0.06-0.05
1294
1: 1.41x108
2: 1.52x108
3:1.72x108
Ave: 1.55x108+0.06-0.05
Recultured with 1-2-1: closest to average
1296: 3.6μl
W303A: 3.2μl
1294: 4.8μl

8/4/14

  • Made one 250mL bottle of liquid YPD media (2%) because of contamination in other bottle
  • Made sporulation media
    potassium acetate (1%)2.5g
    bacto-yeast extract (0.1%)0.25g
    glucose (0.05%)313μL
    H2O250mL
  • Yeast roller stopped overnight, so cell density was comparatively lower than expected for today.
    W303A
    (1) 4.75x107 cells/mL -- 10.53μL for dilution
    (2) 4.05x107 cells/mL -- 12.35μL for dilution
    (3) 4.35x107 cells/mL -- 11.5μL for dilution
    1296
    (1) 4.24x106 cells/mL -- 118μL for dilution
    (2) 4.955x106 cells/mL -- 101μL for dilution
    (3) 4.15x106 cells/mL -- 125μL for dilution
    1294
    (1) 2.715x106 cells/mL -- 184μL for dilution
    (2) 2.315x106 cells/mL -- 216μL for dilution
    (3) 3.02x106 cells/mL -- 165.5μL for dilution
  • The yeast was not rediluted and put back into the roller until 5:40pm.
  • Made plate readings of W303A(1) for OD600
    OD600cell density (cells/mL)
    1.5562x108
    1.0371x108
    0.7955.17x107
    0.6184.78x107
    0.4172.51x107
    0.2361.535x107
    0.1348.35x105
    0.1251.05x105
  • Colony PCR'ed EST2, Mak31, Vps75 for biobricking

For tomorrow: start new yeast experiment(?), continue yeast experiment (5:40 check), autoclave more glass culture tubes, biobrick parts

8/6/14

  • Gave presentation on project to summer STEM participants.
  • Autoclaved culture tubes
  • Checked roller at ~4:15
    W303A
    1. 6.20x107 cells/mL -- 8.06μL for dilution
    2. 5.70x107 cells/mL -- 8.77μL for dilution
    3. 7.03x107 cells/mL -- 7.1μL for dilution
    1296
    1. 1.47x107 cells/mL -- 34.0μL for dilution
    2. 1.99x107 cells/mL -- 25.1μL for dilution
    3. 1.74x107 cells/mL -- 28.7μL for dilution
    1294
    1. 2.33x107 cells/mL -- 21.5μL for dilution
    2. 2.41x107 cells/mL -- 20.7μL for dilution
    3. 2.33x107 cells/mL -- 21.4μL for dilution
  • Put rollers back in for one more day(to complete 7 days)
  • Put 1305 diploid into sporulation media and restruck two plates of 1305.


Tomorrow: Check rollers at 4:30. PCR biobrick parts. prepare psb1c3 vector for biobricking.

8/7/14

  • PCR of Biobrick Parts: VPS75::TRP1 (colony 6), MAK31:: TRP1 (colony B), EST1 (13000)
    1-2kb program with extension time 60sec and annealing temperature 56°C
    Want 500 ng of the vector: psB1C3: 71.1 ng/μl=>7μl
    Biobrick Digest Procedure
    EcoRI Enzyme: 1μl
    PstE Enzyme: 1μl, should be added last.
    1μl of enzyme: 10μl total-> make 20 total but professor suggested 15μl total
    1.5μl BSA, 1.5μl Buffer H, 3μl water =>15μl total
    37°C incubation for 1 hour, 70°C innactivation period 15 min.
  • Purify using PCR product procedure
  • Nanodrop and run on gel (expected size of vector: 2040 bp)
    Final concentration of psB1C3: 9.0 ng/μL
  • Gel Run of PCR'd biobrick parts


    Wells: 1 blank ; 2 Versa Ladder ; 3 EST2 ; 4 MAK31 ; 5 VPS75
    Results
    faint band on 1K for VPS75. Redyed but did not make clearer
  • Put in two samples of 1305 in sporulation media at ~2:30 pm
  • Gel Run of purified psB1C3 vector


    Wells: 1 blank ; 2 Versa Ladder ; 3 psB1c3 purified
    Results
    OD600 of yeast cells
    W303A: 1: 1.161; 2:1.143; 3:1.367
    1294: 1:1.027; 2.1.080; 3:1.079
    1296: 1: 0.987; 2.1.224; 3:1.091


For tomorrow: purify VPS75 biobrick part; double digest VPS75; insert VPS75 part into psB1C3 vector; check cells in sporulation media ; rerun PCR of EST2 and MAK31 with different settings?

8/8/14

  • Redo of Colony PC EST2 and MAK31
    Used Q5 Master Mix with buffers
    12.5μl of 1X Mix
    1.25μl of FWD and RVS Primers
    10μl of template
    25μl reaction total
  • PCR Settings
    Denaturation: 98°C, 30sec
    30Cycles: 98°C, 30sec
    55°C, 30sec
    72°C, 30sec
    Extension Time: 72°C, 2min
    Hold at 4°C
  • Gel Run of EST2 and MAK31
    Well1: blank, 2: Versa Ladder, 3: MAK31, 4: EST2
    Results
    no bands so PCR did not work
  • Nanodrop malfunction
    concentration of purified VPS75 unchecked:
    double digest VPS75 and insertion of VPS75 part into psB1c3 vector not operated
  • Made graph of yeast growth curve (with changed OD600 equation)

For monday: New yeast cycle, check Spo, double digest VPS75; insert VPS75 part into psB1C3 vector;

8/11/14

  • Sporulation media culture
    8/6 culture contaminated
    8/7 cultured for 4 days showed clustering
    1000x KANMX selection
    2.25 ml sterile PBS
    G418 disulfate salt
    filter sterilized and refrigerated
  • 1305 selection plates: RAD52:LEU2//EST2:KANMX//SGS1:HIS3
  • Haploid seletion
  • zymolyase breaks down membrane==>cultured on YPD plates. then replicated onto LEU-, KANMX, HIS3-
  • double selection on LEU, KANMX plates
  • 200ul zymolyase storage buffer to zymolyase 1000U
  • VPS75 purification check: 12.2 ng/μl
  • Made 3 plates each of W303Aα with 35 cells/mL W303Aα 350 cells/mL

Tomorrow: double digest VPS75 insert, biobrick transformation, replicate spo plates, screen EST1+VPS75/MAK31 colonies for double knockouts.

For future: generate growth curve for double selected spo plates (EST2+RAD52) and EST1+VPS/MAK

8/12/14

  • double digested VPS75 insert
    45μL VPS75 (12.2 ng/μL)
    1.5μL each ECOR1 and PST1
    6μL buffer H
    6μL 10x BSA
    60μL total
    Incubated for 1 hour (no heat inactivation)
  • Purified VPS75 insert (Final concentration: 1.1 ng/μL)
    Need to redo PCR. Next time PCR, dilute to 50μL, then directly double digest
  • Inoculated 1296, W303α, VPS75::TRP1 colony 6, MAK31::TRP1 colony B for new curves. Put in roller at 2:45 pm
  • colony PCR screened for double knockouts (EST1 and VPS/MAK)
    A = VPS75::TRP1 ; EST1::LEU2 "Colony 6" Plate 2 (strain 1880)
    B = MAK31::TRP1 ; EST1::LEU2 "Plate B" Plate 2 (strain 1880)
    C = MAK31::TRP1 ; EST1:: LEU2 "Plate B" Plate 1 (strain 1880)
    D = MAK31::TRP1 ; EST1:: LEU2 "Colony 7" Plate 1 (strain 1880)
    E1-E5 = MAK31::TRP1 ; EST1::LEU2 "Colony 3" Plate 2 (strain 1880)
  • PCR conditions:
    Initial: 95°C, 300 sec
    40 cycles
    Denature: 95°C, 30 sec
    Anneal: 56°C, 30 sec
    Extension: 72°C, 60 sec
    Final: 72°C, 420 sec
    Hold: 4°C
  • Made 4 1% gels
    Ran gels of colony PCR check for EST1
    Gel 1 Wells: 1 Versa Ladder ; 2 A3 ; 3 A4 ; 4 B3 ; 5 B4 ; 6 C3 ; 7 C4
    Gel 2 Wells: 1 Versa Ladder ; 2 D3 ; 3 D4 ; 4 E1 ; 5 E2 ; 6 E3 ; 7 E4 ; 8 E5
    Gel Results
  • Checked sporulated yeast cells, dissolved membrane with 10μL zymolyase (30 minute wait)
  • inoculated two tubes of 1305 in Spo media

For tomorrow: Make YPD plates, redo BioBrick of VPS75 (do not purify PCR product), look at primers of EST1 and MAK31 (BioBrick), check sporulated yeast plates (replicate if there is growth), check rollers at 2:45

8/13/14

  • YPD plates made
  • VPS75 colony PCR: From colony 6, 2 samples for biobricking
    Initial: 95°C, 300 sec
    30 cycles
    Denature: 95&Deg;C, 30 sec
    Anneal: 56°C, 30 sec
    Extension: 72°C, 60 sec
    Final: 72°C, 120sec
    Hold: 4°C
  • use 1300 for EST2 biobrick PCR (post senescent)
  • made 2 250 mL bottles for YPD plates
  • Ran gel of VPS75 biobrick (~1K bp expected)


    Gel 1 Wells: 1 Blank ; 2 QuickLoad 1Kb Lab ; 3 colony 6, VPS751 ; 4 VPS75 2 ;
    Results: 1 kB bands
  • PCR of EST2, MAK31 biobrick: EXT time 90 sec, Final step: 420 sec
  • yeast growth curve check: OD600 readings
    YPD blank EST2 neat VPS75 neat MAK31 neat W303A neat
    OD600 .112 .610 1.345 1.318 1.209
    cell density 6.1x107 1.345x108 1.318x108 1.209x108
    dilution volume 8μL 4μL 4μL 4μL

    cells diluted to 1x105cells/μL, triplicate samples, put in roller at 3:30PM
  • Nanodrop concentrations
    VPS751 527.0 ng/μL
    VPS752 690.9 ng/μL

For tomorrow: double digest VPS75 insert
Redo PCRs for double knockout screening
Prepare for meeting
Check yeast rollers
Check sporulated yeast plates

8/14/14

  • Double Digest: (done for each VPS75 1 and VPS752)
    Buffer H: 2μL
    BSA: 2μL
    VPS75: 3.7μL
    ddH2O: 10.3μL
    EcoRI: 1μL
    PstI: 1μL
    Incubate at 37°C for 60 min
    Heat at 70°C for 15 min
  • Checked concentrations, but received negative concentrations... did calculations manually:
    VPS751: 2556.33 ng/20μL = 127.8 ng/μL
    VPS752: 1950 ng/20μL = 97.5 ng/μL
  • Stored double digested VPS751 and VPS752 at -20°C till further instructions on how to ligate.
  • Ran gel of yesterday's PCR to check biobrick primers
    Wells: 1, blank; 2, versa ladder; 3, E; 4, MB; 5, M7
  • Made four 1% gels
  • colony PCR'ed 13 samples for double knockout (Program 334)
    PCR conditions
    ext. time 30
    anneal temp 56&Deg;C
    final step 420
    cycles 30
  • yeast growth curve check: OD600 readings
      Blank YPD MAK31 1 MAK31 2 MAK31 3 EST2 1 EST2 2 EST2 3 VPS75 1 VPS75 2 VPS75 3 W303A 1 W303A 2 W303A 2
    OD600 0.116 1.246 1.22 1.084 0.178 0.242 0.222 1.219 1.192 1.189 1.128 1.127 1.037
    Dilution Vol 4μL 4μL 4.6μL 28μL 20.7μL 22.5μL 4.1μL 4.2μL 4.2μL 4.4μL 4.4μL 4.8μL

    put yeast cultures in roller @ 4:00PM
  • Duplicated 3 plates of sporulated cells onto
    Leu-/KANMX (Rad52::Leu2 and EST2::KANMX)
    Leu- (RAD52::LEU2)
    KANMX (EST2::KANMX)
    HIS- (SGS1::HIS3)
  • Updated wiki outreach page

8/15/14

  • Observed 1 cell with RAD52 and EST2 knocked out as well as 1 cell with EST2 and SGS1 knocked out
  • the Leu-/KANMX plate may be missing the EST2::KANMX since those plates look identical to the Leu- Plates


    Gel 1: 1, Versa Ladder; 2, A3; 3, A4; 4, B3; 5, B4; 6, C3; 7, C4; 8, E3;
    Gel 2: 1, Versa Ladder; 2, D3; 3, D5; 4, E1; 5, E2; 6, E3; 7, E4; 8, E5;

    The E3 in Gel 2 looked faint so we ran E3 on gel 1 as well.
  • Found protocol to isolate DNA in yeast cells
    RAD52 EST2 Knockouts
    inoculate culture (5 mL for curve)
    use 1 mL of saturated culture
    mix above with 1 mL of 60% glycerol
    vortex (label clearly)
    store at -80°C
    RAD52 EST2 Growth Curve
    Take 1/2 of colony
    inoculate in 5 mL YPD
    triplicate samples tomorrow
  • Make more sporulated yeast cell plates (~500 cells)
    Zymolased with 10μL
    Made 6 plates with ~450 cells
  • Yeast Growth Curves
      YPD W303A 1 W303A 2 W303A 3 MAK31 1 MAK32 2 MAK32 3 VPS 1 VPS 2 VPS 3 EST2 1 EST2 2 EST2 3
    OD600 .146 1.074 0.937 0.994 1.035 1.250 1.250 1.184 1.187 1.044 0.762 1.321 0.469
    Dilution 4.6μL 5.3μL 5μL 4.8μL 4μL 4μL 4.2μL 4.2μL 4.8μL 6.6μL 3.8μL 10.7μL
  • Also made a RAD52 & EST2 tube and put in roller at ~5:30PM

8/16/14

Yeast Growth Curves
  YPD W303A 1 W303A 2 W303A 3 VPS 1 VPS 2 VPS 3 EST2 1 EST2 2 EST2 3 MAK31 1 MAK32 2 MAK32 3 RAD52&EST2
OD600 .134 .602 .592 .555 .520 .675 .719 .497 .681 .582 1.032 .920 1.008 0.836
Dilution 8μL 8μL 8μL 7μL 7μL 7μL 7.5μL 7.5μL 7.5μL 5μL 5μL 5μL 6μL

Dilutions
W303A: ~8μL-> 1
VPS75: ~7μL -> 3
EST2: ~7.5μL -> 3
MAK31: ~5μL -> 1
RAD52: ~6μL

Sporulated 2 & 3 were empty and trashed

8/17/14

Yeast Growth Curves
  YPD W303A 1 W303A 2 W303A 3 VPS 1 VPS 2 VPS 3 EST2 1 EST2 2 EST2 3 MAK31 1 MAK32 2 MAK32 3 RAD52&EST2 1 RAD52&EST2 RAD52&EST2 1
OD600 .117 .670 .643 .662 .744 .705 .724 .706 1.510 1.290 1.184 1.207 1.171 .121 .116 .107
.Dilutions 7.5μL 7.8μL 7.6μL 6.7μL 7.1μL 6.9μL 7.1μL 3.3μL 3.9μL 4.2μL 4.1μL 4.3μL 6μL
  • Incubated another RAD52 & EST2 in 5 mL YPD
  • Put in roller @ 5:30PM
  • For tomorrow: freeze saturated RAD52&EST2, make 60% glycerol

8/18/14

Yeast Growth Curves
  W303A (1) W303A (2) W303A (3) EST2(1) EST2(2) EST2(3) MAK31(1) MAK31(2) MAK31(3) VPS75 (1) VPS75 (2) VPS75 (3) RAD52 EST2 (1) RAD52 EST2 (2) RAD52 EST2 (3) RAD52 EST2 (8/17)
OD600 .230 0.298 0.225 0.518 .678 .528 1.196 1.194 1.212 1.115 1.084 1.059 1.018 .137 1.116 .498
Dilution Vol (μL) 21.7 16.8 22.2 9.7 7.4 9.5 4.2 4.2 4.1 4.5 5.0 4.7 4.9 4.5 10

  • Put in roller at 5:00pm
  • Wildtype culture samples were thrown out because of evidence of contamination
  • Ran gel for biobrick primers. Yielded results of ~200bp when the expected result was ~1000bp
  • Made triplicate samples of RAD52 EST2 inoculated yesterday (8/17). Also froze -80°C with 1 mL yeast, 1mL 60% glycerol (2 samples).

8/19/14

  • Zymolased sporulated samples w/ 15μL @ 1:05 PM - 2 tubes - followed protocol from 8/11
  • Sporulated samples turned out to be contaminated, and were thrown out
  • Double Digested VPS75 1 and VPS 75 2
    Incubated @ 1:13PM 37&Deg;C for 60 min
    Heated @ 2:14PM 70°C for 15 min
    followed same protocol for 8/14 double digest, total volume 20μL
  • Purified VPS75 double digests (1 and 2)
    Nanodrop results
    (1): 5.6 ng/μL
    (2): 4.2 ng/μL
  • Made two sporulation tubes and put in roller (Check in 5 days)
    Used 5 mL sporulation media
    1305 (1) and 1305 (2)
  • Yeast Growth Curves
    YPD (blank) VPS(1) VPS(2) VPS(3) MAK31(1) MAK31(2) MAK31(3) EST2(1) EST2(2) EST2(3) RAD52 EST2 (1) RAD52 EST2 (2) RAD52 EST2 (3) RAD52 EST2 1 (8/17) RAD52 EST2 2 (8/17) RAD52 EST2 3 (8/17)
    OD600 .119 1.137 1.130 1.206 1.166 1.190 1.176 1.201 0.570 0.573 0.111 0.546 0.114 0.114 0.121 0.117
    Dilution 4.4μL 4.4μL 4.1μL 4.3μL 4.2μL 4.3μL 4.2μL 8.8μL 8.7μL 9.2μL
  • Need to start generating curves for RAD52/EST2 as well as update graphs and std. dev./error bars

8/20/14

  • Double-knockout Screening: A5, A6, B5, B6, C5, C6, D5, D6, E1, E2, E3, E4, E5. Used same plates as before, choose new colonies for plates A, B, C, and D
  • swirled colonies each in 200μL ddH2O tubes
  • PCR tubes: 20μL ddH2O+colonies, 2.5μL reverse and forward primers (EST1)rs
  • Ligation notes:
  • Team page wiki ideas:
  • Yeast Growth Curves
    YPD EST2 1 EST2 2 EST2 3 MAK31 1 MAK31 2 MAK31 3 VPS75 1 VPS75 2 VPS75 3 RAD52/EST2 1 RAD52/EST2 2 RAD52/EST2 3 RAD52/EST2 1 (START 8/17) RAD52/EST2 2 (START 8/17) RAD52/EST2 3 (START 8/17
    OD600 0.125 1.476 1.121 .609 1.274 1.227 1.259 1.241 1.213 1.206 .107 .458 .109 .129 .142 .143
    Dilution 3.4μL 4.5μL 8.2μL 3.9μL 4.1μL 4.0μL 4.0μL 4.1μL 4.1μL

8/21/14:

  • Made two 1% gels
  • Ran gels of double knockout colony PCR screenings


    Gel 1: VersaLadder, A5, A6, B5, B6, C5, C6
    Gel 2: VersaLadder, D5, D6, E1, E2, E3, E4, E5
  • yeast growth curve
    VPS75 1 VPS75 2 VPS75 3 EST2 1 EST2 2 EST2 3 MAK31 1 MAK31 2 MAK31 3 RAD52/EST2 1 RAD52/EST2 2 RAD52/EST2 3 RAD52/EST2 1 (START 8/17) RAD52/EST2 2 (START 8/17) RAD52/EST2 3 (START 8/17)
    OD600 (x108 cells/mL) 1.150 1.152 1.161 1.265 0.651 0.576 1.147 1.158 1.187 0.101 0.478 1.393 0.140 0.562 0.166
    Dilution (μL) 4.3 4.3 4.3 4 7.7 8.7 4.4 4.3 4.2 10.3 3.6 8.9

8/22/14


Yeast Growth Curve
YPD VPS75 1 VPS75 2 VPS75 3 EST2 1 EST2 2 EST2 3 MAK31 1 MAK31 2 MAK31 3 RAD52/EST2 1 RAD52/EST2 2 RAD52/EST2 3 RAD52/EST2 1 (START 8/17) RAD52/EST2 2 (START 8/17) RAD52/EST2 3 (START 8/17)
OD600 .144 1.154 1.141 1.153 1.274 .655 .666 1.232 1.190 1.197 .105 1.210 1.611 .186 .700 .233
Dilution (μL) 4.3 4.4 4.3 3.9 7.6 7.5 4.1 4.2 4.2 -- 4.1 3.1 -- 7.1 21.5

8/25/14


  • Dephysophorylated vector DNA
  • Ligation calculator 3:1
  • Not Dephosphorylated
    45ng insert DNA - 9μL (5.6 ng/μL)
    30ng vector DNA - 3μL
    10X T4 DNA ligase buffer - 2μL
    water - 5μL
    T4 DNA ligase - 1μL
    Total Volume: 20μL
  • Dephosphorylated
    45ng insert DNA - 9μL (5.6 ng/μL)
    30ng vector DNA - 10μL
    10x T4 DNA ligase buffer - 2.5μL
    water - 2.5vL
    T4 DNA ligase - 1.25μL
    Total Volume: 25μL
  • 15μL zymolase w sporulated cells (protocol on 8/11/14)
  • Counted cells and averaged for 444 in sample 2 and 560 in sample 1
  • Made 3 plates of each sample
  • RAD52/EST2 cells from the last growth curve were contaminated with bacteria
  • Made 2 bottles of 250 mL YPD
    • 1 bottle for YPD plates
    LB plates:
    LB - Lennox Broth - 4g
    agar 1.5% - 3g (in 200 mL)
    Autoclave -> don't add things when it's too hot
    Add Chlor (1000x) - 200 uL​
    40% Glucose
    multiply 40% by mL to get grams of dextrose
    used 120g dextrose and 300 mL
    stirred & heated glucose in 100 mL dH2O then added more dH2O till the desired level of 300mL
  • restreaked MATA and MATα for mating type checks of 1294, 1296, 1300, 1880 (VT6, MTB, MT7)

For tomorrow/things to do
  • if start yeast curves, start them at noon
  • finish up ligation
  • check on plates in incubator - maybe redo kan- plate w/ ypd if we do replicate yeast
  • double knockout screening -check other colonies/re-do
  • biobrick primers
  • yeast dna protocol - redo w/ biobrick primers
  • innoculate RAD52/EST2 colony at noon
  • put ligations in -20°C in morning

    • 8/26/14

    • ligations put in -20°C
    • restreaked MAK31::TRP1 colony 7
    • inoculated and put in roller at noon:
      1880
      VPS75::TRP1 colony 6
      EST2 1296
      RAD52/EST2
    • yeast genomic DNA extraction protocol for biobricking
      nanodrop results
      EST2 (1300) : 41/6 ng/μL
      MAK31 (MT7) : 14.4 ng/μL
      VPS75 (VT6) : 26.0 ng/μL
    • ran PCR of EST2, MAK31, VPS75 for biobricking
      30 cycles
      95°C, 30 sec
      56°C, 30 sec
      72°C, 90 sec
      72°C, 120 sec
    • Made 2 250 mL bottles for YPD plates
    • 1 250 mL bottle for KANMX plates (250μL G418)

    For tomorrow
  • Run gels of biobrick inserts
  • if they worked, double digest, purify
  • transform along with VPS75
  • Select more sporulated cells
  • Check cells growing on sporulated plates
  • Growth curve at noon (make triplicate samples)
  • check mating type of EST2/RAD52, 1294, 1296, 1300, 1880 (VT6, MTB, MT7)
  • mate EST2 and MAK31/VPS75 for sporulation

    • Research
  • gibson assembly (isothermal) for "super plasmid"
  • yeast operons ( for EST2, RAD52 )
  • prerecombinase done in iGEM already?
  • biobuilder.org -> look for beta carotene yeast, possibly use as a measure to characterize growth curves, senescence vs nonsenecence
  • thin layer chromatography (TLC)
  • gas chromatography
  • check iGEM deadlines

    • 8/27/14


    • Zymolased (2) and (3) sporulation samples with 15μL at 11:30 AM, wait 30 min
      (2) after diluting 2 times 1/10, counted an average of 88 cells (with 87 and 95)
      (3) after diluting 2 times 1/10, counted on average 82 cells (with 80 and 84)
    • Made 2 1% gels
      Ran gel of biobricking PCR product

      Wells: 1 blank ; 2 blank ; 3 Versa Ladder ; 4 EST2 ; 5 MAK31 ; 6 VPS75
      Gel Results
      EST2 - band on 2K bp
      MAK31 - band on 600 bp
      VPS75 -band on 1K bp
      1K expected...
    • checked mating types of 1880, VT6, MTB, MT7, EST2/RAD52
    • Made 1 250 mL bottle for SD plates (for mating selection) (no dropout mix added
      1 bottle KAN/LEU
      1 bottle HIS

    8/29/2014


    • Note: 1880 is DSY strain
    • 2 250 ml LEU- plates made
    • VPS75 BB transformation check
      1 white colony on 1 dephosphorylcoted plate
      dense reds on not plates==> restreaked white colonies on gridded chlor- plates
    • Mating type plate of EST2/RAD52, 1880, VT6, MTB,MT7
      =>MATA:1880 MTB,VT6,EST2,RAD52
      MT7 grew on both sides (possible diploid)
    • replicated 1294 1296 1300 on SD selection plate
    • replicated sporulated cells
      Stopped growth curve for weekend
      Cell densities in notebook



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