Difference between revisions of "20.109(F16): TA notes for M1"

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{{Template:20.109(F16)}}
  
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For Hsinhwa
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<div style="padding: 10px; width: 790px; border: 5px solid #000FFF;">
  
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Module 1 Measuring Genomic Instability
+
===M1D1===
−
Number of groups: 9, so prepare 11 aliquots
+
Per group, aliquot
 +
*50 mL 1x PBS
 +
*in TC room, 15 mL of “TK6 media”
  
−
Week 1: September 12, 2016
+
===M1D2===
−
Day 1 (T/W)
+
Per group, aliquot
−
Per group:
+
*in main lab
−
• Aliquot 50 mL 1x PBS per group (label: PBS)
+
**10 mL 1x PBS  
−
• In TC room, aliquot 15 mL of “TK6 media” (label: TK6 media)
+
**2 eppendorf tubes of 0.3% LMP agarose (in eppendorf water bath)
 +
**large scheme of the plate so they can keep track of how to load chips
 +
*and separately, in the TC hood,  
 +
**10 mL 1x PBS
 +
**25 mL TK6 media
 +
**2 eppendorf tubes of 0.3% LMP agarose (in water bath '''at 43 &deg;C''')
 +
**2x 15 mL of TK6 cells at 500,000 cells/mL
 +
Set up hoods in TC room with, per group:
 +
*4 binder clips
 +
*1 glass slide
 +
*1 bottomless 96-well plate
 +
*1 razor blade
 +
*Pasteur pipets
 +
*sharp jar
 +
*pipet aid
 +
*pen
 +
*team sticker to identify glass slides
 +
*printout of today's protocol
 +
*large scheme of the plate to hang in TC so they can keep track of how to load chips
  
−
Day 2 (R/F)
+
===M1D3===
−
Per group:
+
In incubator, have (per group)
−
• Aliquot 10 mL 1x PBS (label: PBS)
+
*TK6 cells at 500,000 cells/mL (10 mL)
−
• And separately, in the TC hood, aliquot 10 mL 1x PBS (label: PBS)
+
−
Leslie & Maxine will prepare:
+
−
• Prepare 4 x 2 mL tubes of 0.3% LMP agarose, per group
+
  
−
Week 2: September 19, 2016
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On front bench, prepare (per group)
−
Day 3 (T/W)
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*razor blades (1)
−
Leslie & Maxine will prepare:
+
*bottomless 96-well plates (3)
−
• 1% LMP agarose
+
*glass slides (2)
−
• H2O2 and MMS
+
*binder clips (8)
−
• Alkyline lysis solution
+
*water bath at 43 &deg;C with
−
• Alkyline electrophoresis buffer
+
*1% LMP agarose (3 mL)
−
• Neutralization buffer
+
*12-well reservoirs (1)
−
• SYBR Gold buffer
+
*multi-channel 300 &mu;L pipets (1)
 +
*boxes for lysis incubation (1 dead, 1 live)
 +
*lysis buffer stock (40 mL)
 +
*Triton X-100 (400 &mu;L)
 +
*Neutralization buffer (50 mL)
 +
*SYBR Gold buffer (40 mL)
  
−
Week 3: September 26, 2016
+
In cold room, prepare (per group)
−
Day 4 (T/W)
+
*electrophoresis gel boxes with double-sided tape (2 teams or 4 CometChips per box)
−
Per group:
+
*alkyline electrophoresis buffer (500 mL)
−
• Aliquot 15 mL 1x PBS (label: PBS)
+
−
Leslie & Maxine will prepare:
+
−
• H2O2
+
−
• Alkyline lysis solution
+
−
• Warm culture medium
+
  
 +
For H2O2, on each team's bench, prepare
 +
*ice bucket with
 +
*12-well reservoir
 +
*50 mL PBS
 +
*H2O2 at 10 mM in cold PBS
  
−
Day 5 (R/F)
+
For MMS, on front bench, prepare (per group)
−
Per group:
+
*M and L flocked-lined gloves
−
In TC room for all aliquots, leave at 4C:
+
*MMS at 80 mM (1.25 mL RPMI + 8.5 &mu;L of 12 M stock, prepared in fume hood)
−
• Aliquot 5 mL of 0.1% gelatin (label: Gelatin)
+
*RPMI1640 medium (12 mL)
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• Aliquot 10 mL of PBS (label: PBS)
+
*PBS (20 mL)
−
• Aliquot 3 mL of 1X trypsin (label: Trypsin)
+
*dedicated MMS liquid waste basins
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• Aliquot 15 mL of M059J/K media (label: Media)  
+
−
Leslie & Maxine will prepare:
+
−
• M059J/K in T25 flasks
+
  
−
Week 4: October 3, 2016
+
For MMS, aliquot (per team)
−
Day 6 (T/W)
+
*TK6 media
−
Per group:
+
*MMS at 80 mM (250 &mu;L)
−
• Aliquot 10 mL of 1x TBS (label: TBS)
+
−
• Aliquot 500 uL of 10% BSA, frozen stock in -20C (label: 10% BSA)
+
−
Leslie & Maxine will prepare:
+
−
• 10% BSA and freeze at -20C
+
−
• Triton X-100 at front bench
+
−
• Primary antibodies
+
  
−
Day 7 (R/F)
+
===M1D4===
−
Per group:
+
In incubator, have (per group)
−
• Aliquot 5 mL of 1x TBS (label: TBS)
+
*Coriell # 10 (300,000 cells)
 +
*Coriell # 20 (300,000 cells)
 +
*Coriell # 24 (300,000 cells)
 +
 
 +
Per group, aliquot:
 +
*25 mL 1x PBS
 +
*25 mL RPMI 1640 media (non sterile, warm in incubator)
 +
*liquid trash basin
 +
 
 +
On front bench, prepare (per team)
 +
*razor blades (1)
 +
*glass slides (1)
 +
*binder clips (4)
 +
*bottomless 96-well plate (1)
 +
*1% LMP agarose (2 mL)
 +
*water bath at 43 &deg;C
 +
*'''cold''' H2O2 at 10 mM (10 mL PBS + 10 &muL H2O2 10 M stock; each team needs < 100 &mu;L)
 +
*reservoir (1)
 +
*multichannel pipet (1)
 +
*tray boxes (1 + 1 for lysis and for recovery)
 +
*lysis stock buffer (30 mL)
 +
*Triton X-100 (300 &mu;L)
 +
 
 +
Tomorrow, will need (per team)
 +
*Neutralization buffer (75 mL)
 +
*SYBR Gold buffer (40 mL)
 +
*electrophoresis gel boxes with double-sided tape (2 teams or 2 CometChips per box)
 +
*alkyline electrophoresis buffer (500 mL)
 +
 
 +
===M1D5===
 +
Per group, aliquot in TC room:
 +
*5 mL of 0.1% gelatin
 +
*10 mL of PBS
 +
*3 mL of 1X trypsin
 +
*15 mL of M059J/K media
 +
*M059J/K in T25 flasks
 +
 
 +
===M1D6===
 +
Per group, aliquot:
 +
*5 mL TBS
 +
*5 mL blocking buffer (4.5 mL of TBS + 500 &mu;L of 10% BSA, frozen stock in -20C)
 +
*700 &mu;L blocking buffer
 +
*one staining chamber
 +
*one gauge 26 needle
 +
*one pair of tweezers
 +
 
 +
On front bench, prepare:
 +
*Triton X-100
 +
*Primary antibodies
 +
 
 +
===M1D7===
 +
Per group, aliquot
 +
*1.5 mL of 1x TBS
 +
*4 microscope slides
 +
*1 gauge 26 needle
 +
 
 +
On front bench, prepare
 +
*ProLong Gold anti-fade reagent with DAPI
 +
*beaker of water
 +
*nail polish
 +
*book to transport slides

Latest revision as of 13:14, 6 October 2016

20.109(F16): Laboratory Fundamentals of Biological Engineering

Engelward PNAS 2006.png

Schedule Fall 2016        Announcements        Assignments        Homework        Communication
       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

M1D1

Per group, aliquot

  • 50 mL 1x PBS
  • in TC room, 15 mL of “TK6 media”

M1D2

Per group, aliquot

  • in main lab
    • 10 mL 1x PBS
    • 2 eppendorf tubes of 0.3% LMP agarose (in eppendorf water bath)
    • large scheme of the plate so they can keep track of how to load chips
  • and separately, in the TC hood,
    • 10 mL 1x PBS
    • 25 mL TK6 media
    • 2 eppendorf tubes of 0.3% LMP agarose (in water bath at 43 °C)
    • 2x 15 mL of TK6 cells at 500,000 cells/mL

Set up hoods in TC room with, per group:

  • 4 binder clips
  • 1 glass slide
  • 1 bottomless 96-well plate
  • 1 razor blade
  • Pasteur pipets
  • sharp jar
  • pipet aid
  • pen
  • team sticker to identify glass slides
  • printout of today's protocol
  • large scheme of the plate to hang in TC so they can keep track of how to load chips

M1D3

In incubator, have (per group)

  • TK6 cells at 500,000 cells/mL (10 mL)

On front bench, prepare (per group)

  • razor blades (1)
  • bottomless 96-well plates (3)
  • glass slides (2)
  • binder clips (8)
  • water bath at 43 °C with
  • 1% LMP agarose (3 mL)
  • 12-well reservoirs (1)
  • multi-channel 300 μL pipets (1)
  • boxes for lysis incubation (1 dead, 1 live)
  • lysis buffer stock (40 mL)
  • Triton X-100 (400 μL)
  • Neutralization buffer (50 mL)
  • SYBR Gold buffer (40 mL)

In cold room, prepare (per group)

  • electrophoresis gel boxes with double-sided tape (2 teams or 4 CometChips per box)
  • alkyline electrophoresis buffer (500 mL)

For H2O2, on each team's bench, prepare

  • ice bucket with
  • 12-well reservoir
  • 50 mL PBS
  • H2O2 at 10 mM in cold PBS

For MMS, on front bench, prepare (per group)

  • M and L flocked-lined gloves
  • MMS at 80 mM (1.25 mL RPMI + 8.5 μL of 12 M stock, prepared in fume hood)
  • RPMI1640 medium (12 mL)
  • PBS (20 mL)
  • dedicated MMS liquid waste basins

For MMS, aliquot (per team)

  • TK6 media
  • MMS at 80 mM (250 μL)

M1D4

In incubator, have (per group)

  • Coriell # 10 (300,000 cells)
  • Coriell # 20 (300,000 cells)
  • Coriell # 24 (300,000 cells)

Per group, aliquot:

  • 25 mL 1x PBS
  • 25 mL RPMI 1640 media (non sterile, warm in incubator)
  • liquid trash basin

On front bench, prepare (per team)

  • razor blades (1)
  • glass slides (1)
  • binder clips (4)
  • bottomless 96-well plate (1)
  • 1% LMP agarose (2 mL)
  • water bath at 43 °C
  • cold H2O2 at 10 mM (10 mL PBS + 10 &muL H2O2 10 M stock; each team needs < 100 μL)
  • reservoir (1)
  • multichannel pipet (1)
  • tray boxes (1 + 1 for lysis and for recovery)
  • lysis stock buffer (30 mL)
  • Triton X-100 (300 μL)

Tomorrow, will need (per team)

  • Neutralization buffer (75 mL)
  • SYBR Gold buffer (40 mL)
  • electrophoresis gel boxes with double-sided tape (2 teams or 2 CometChips per box)
  • alkyline electrophoresis buffer (500 mL)

M1D5

Per group, aliquot in TC room:

  • 5 mL of 0.1% gelatin
  • 10 mL of PBS
  • 3 mL of 1X trypsin
  • 15 mL of M059J/K media
  • M059J/K in T25 flasks

M1D6

Per group, aliquot:

  • 5 mL TBS
  • 5 mL blocking buffer (4.5 mL of TBS + 500 μL of 10% BSA, frozen stock in -20C)
  • 700 μL blocking buffer
  • one staining chamber
  • one gauge 26 needle
  • one pair of tweezers

On front bench, prepare:

  • Triton X-100
  • Primary antibodies

M1D7

Per group, aliquot

  • 1.5 mL of 1x TBS
  • 4 microscope slides
  • 1 gauge 26 needle

On front bench, prepare

  • ProLong Gold anti-fade reagent with DAPI
  • beaker of water
  • nail polish
  • book to transport slides
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