Difference between revisions of "20.109(F16): TA notes for M2"

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Fa16: all done by 4:30pm
 
Fa16: all done by 4:30pm
 
'''Should schedule BE Comm Lab workshop on Journal Club on this day.'''
 
'''Should schedule BE Comm Lab workshop on Journal Club on this day.'''
 +
 +
===M2D3===
 +
On ice,
 +
*1.2 mL aliquots of nuclease-free water
 +
*reverse primer at 100 μM
 +
*psgRNA template
 +
*HotStart Master Mix
 +
*-20 °C block for PCR tubes
 +
*strip of PCR tubes with colors (+1 for control)
 +
*SDM control primer mix
 +
*SDM control plasmid
 +
 +
Also on front bench,
 +
*filtered tips
 +
*gRNA forward primers received from IDT
 +
 +
The thermocycling program is called '''NEBSDM'''.
 +
 +
Later, on ice
 +
*KLD reaction buffer
 +
*KLD enzyme mix
 +
*NEB5α cells (1 per group + 1 for control)
 +
*LB+Amp plates (1 per group + 1 for control)
 +
 +
===M2D5===
 +
The day before lab (8pm), inoculate 2 colonies per team.
 +
 +
Aliquot, per team:
 +
*2x 5 mL cultures of NEB5α transformed with pgRNA and cultured in LB+Amp
 +
*ice bucket
 +
*15 mL conical tube, empty, labeled 'Qiagen waste'
 +
*P1 (600 μL)
 +
*P2 (600 μL)
 +
*P3 (800 μL)
 +
*PB(1100 μL)
 +
*PE (1600 μL) of buffer PE
 +
*water pH 8.0 for elution (50 μL)
 +
*2 mini-prep columns
 +
*MG1655 competent cells (100 μL), '''on ice''' late in the afternoon
 +
*SOC (1 mL)
 +
*2 LB agar plates +Amp+Cam
 +
*burner + lighter + spreader + jar of ethanol
 +
 +
On front bench,
 +
*students plates of pgRNA
 +
*water bath at 42 °C
 +
*sequencing F and R primers at 5 μM
 +
*nuclease-free water
 +
*PCR tube strips for sequencing
 +
 +
===M2D7===
 +
Aliquot, per team:
 +
*LB broth (45 mL)
 +
*chloramphenicol (50 μL, from stock in -20 °C freezer)
 +
*ampicillin (50 μL, from stock in 4 °C deli fridge)
 +
*glass culture tubes (4)
 +
*15 mL conical tubes (4)
 +
 +
===M2D8===
 +
On front bench, per team:
 +
*cuvettes for spectrophotometer (9)
 +
*conical tubes (4)
 +
*eppendorf tubes (16)
 +
*on ice: for kits
 +
**standard
 +
**substrate
 +
**enzyme
 +
*aluminum foil
 +
 +
Aliquot, per team
 +
*LB (20 mL for ethanol, 10 mL for lactate)
 +
*PBS (none for ethanol, 4 mL for lactate)
 +
*assay buffer (4.5 mL for ethanol, 4 mL for lactate)

Latest revision as of 18:49, 8 November 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              

M2D1

Per group, aliquot

  • 30 μL nuclease-free water
  • 5 μL pdCas9 (ideally at 500 ng/μL)

On front bench, have

  • restriction enzymes in -20 °C block
  • NEB buffers
  • list of 20.109-owned restriction enzymes

Fa16: all done by 5pm

M2D2

Per group,

  • pour 1/2 agarose gel (only 5 lanes needed)
  • thaw 4 samples of confirmation digests
  • aliquot 10 μL of 6X loading buffer
  • thaw 20 μL of 1 kb ladder

Call IDT to request expedited FedEx shipping of gRNA oligos.

Fa16: all done by 4:30pm Should schedule BE Comm Lab workshop on Journal Club on this day.

M2D3

On ice,

  • 1.2 mL aliquots of nuclease-free water
  • reverse primer at 100 μM
  • psgRNA template
  • HotStart Master Mix
  • -20 °C block for PCR tubes
  • strip of PCR tubes with colors (+1 for control)
  • SDM control primer mix
  • SDM control plasmid

Also on front bench,

  • filtered tips
  • gRNA forward primers received from IDT

The thermocycling program is called NEBSDM.

Later, on ice

  • KLD reaction buffer
  • KLD enzyme mix
  • NEB5α cells (1 per group + 1 for control)
  • LB+Amp plates (1 per group + 1 for control)

M2D5

The day before lab (8pm), inoculate 2 colonies per team.

Aliquot, per team:

  • 2x 5 mL cultures of NEB5α transformed with pgRNA and cultured in LB+Amp
  • ice bucket
  • 15 mL conical tube, empty, labeled 'Qiagen waste'
  • P1 (600 μL)
  • P2 (600 μL)
  • P3 (800 μL)
  • PB(1100 μL)
  • PE (1600 μL) of buffer PE
  • water pH 8.0 for elution (50 μL)
  • 2 mini-prep columns
  • MG1655 competent cells (100 μL), on ice late in the afternoon
  • SOC (1 mL)
  • 2 LB agar plates +Amp+Cam
  • burner + lighter + spreader + jar of ethanol

On front bench,

  • students plates of pgRNA
  • water bath at 42 °C
  • sequencing F and R primers at 5 μM
  • nuclease-free water
  • PCR tube strips for sequencing

M2D7

Aliquot, per team:

  • LB broth (45 mL)
  • chloramphenicol (50 μL, from stock in -20 °C freezer)
  • ampicillin (50 μL, from stock in 4 °C deli fridge)
  • glass culture tubes (4)
  • 15 mL conical tubes (4)

M2D8

On front bench, per team:

  • cuvettes for spectrophotometer (9)
  • conical tubes (4)
  • eppendorf tubes (16)
  • on ice: for kits
    • standard
    • substrate
    • enzyme
  • aluminum foil

Aliquot, per team

  • LB (20 mL for ethanol, 10 mL for lactate)
  • PBS (none for ethanol, 4 mL for lactate)
  • assay buffer (4.5 mL for ethanol, 4 mL for lactate)
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