20.109(S14): TA notes for module 2

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'''Materials required:'''  
'''Materials required:'''  
Please read as much as you can of Parts 1-3 and create an automated worksheet (e.g., in Excel) that will perform the required calculations for the day. A partially prepared worksheet is [[Media: S14_M2D2-starter.xlsx | '''linked here''']]. We will NOT collect your final worksheet this time; it is only for your benefit.
Please read as much as you can of Parts 1-3 and create an automated worksheet (e.g., in Excel) that will perform the required calculations for the day. A partially prepared worksheet is [[Media: S14_M2D2-starter.xlsx | '''linked here''']]. We will NOT collect your final worksheet this time; it is only for your benefit.
 +
 +
Prep. For westerns:
 +
Make aliquots:
 +
Chill scrapers
 +
RIPA lysis buffer
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Protease inhibitor (last minute)
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Eppendorf tubes
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PBS for wash
 +
Ice buckets
 +
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Set up boiling station:
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Turn on hot plates with water in glass bowl with boiling chips
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Aliquot beta-mercap.
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Don't forget to show students the cap covers
 +
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Set up westerns:
 +
Make sds running buffer
 +
Make transfer buffer (chilled)
 +
Set up gels(remove strips, place in boxes, remove combs, wash wells with running buffer)
 +
Night before: freeze ice blocks
 +
 +
Presoak filter paper and scotch pads in transfer buffer for 30 min prior to blot.
 +
 +
Make sure we have blocking buffer.
 +
'''Day of Lab (R/F):'''
'''Day of Lab (R/F):'''

Revision as of 16:02, 22 March 2014

20.109(S14): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2014        Assignments       
Module 1        Module 2        Module 3              

Contents

General notes

I need to figure out the quiz dates.

See also GoogleDoc for aliquoting amounts.

Scheme: Each pair of students will test K1 and xrs6 cells for Ku80 production. They will then assess the ability of K1, xrs6, and DNA-PKcs inhibited cells to repair DNA damage via plasmid based assay and flow cytometry.

Key preparation:

Cells:

  • Prepare T25 flasks of K1 and xrs6 for every team

Westerns:

  • Prepare transfer buffer

DNA plasmids:

  • Aliquot plasmids

Lipofection: Flow cytometry:


Note to TA:

  • While you read the wiki, test each link, and make any link descriptions bold that are not already.
  • As you read the protocols, add reagent calculations to the GoogleDoc as needed.

Day-by-day

Day 1

Materials required:

1. Aliquots of PBS, CHO media, and 0.25% Phenol Red Trypsin for each group 2. T25 flask of K1 and xrs6 cells for each group

  • Flasks for next day seeded at 600,000 cells
  • Flasks for day after next, seeded at 400,000 cells
  • Flasks for 3 days in future, seeded at 200,000 cells

Day of Lab (T/W):

  • No Quiz
  • Prepare remaining aliquots for students
  • Warm up media, PBS, and trypsin half hour before students use reagents

Additional Prep.:

  • Digest pMAX-BFP-2MCS2 for testing damage topologies
  • Pour gel for purification

After Lab:

  • Aliquot new shipment of trypsin

How it went:

Day went pretty smoothly; not many questions on Part 4 exercise.

Day 2

Materials required: Please read as much as you can of Parts 1-3 and create an automated worksheet (e.g., in Excel) that will perform the required calculations for the day. A partially prepared worksheet is linked here. We will NOT collect your final worksheet this time; it is only for your benefit.

Prep. For westerns: Make aliquots: Chill scrapers RIPA lysis buffer Protease inhibitor (last minute) Eppendorf tubes PBS for wash Ice buckets

Set up boiling station: Turn on hot plates with water in glass bowl with boiling chips Aliquot beta-mercap. Don't forget to show students the cap covers

Set up westerns: Make sds running buffer Make transfer buffer (chilled) Set up gels(remove strips, place in boxes, remove combs, wash wells with running buffer) Night before: freeze ice blocks

Presoak filter paper and scotch pads in transfer buffer for 30 min prior to blot.

Make sure we have blocking buffer.


Day of Lab (R/F):

How it went:

Day 3

Materials required:


Day of Lab (T/W):

After Lab:

How it went:

Day 4

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 5

Materials required:

Day of Lab (T/W):

After Lab:

How it went:

Day 6

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 7

Materials required:

Day of Lab (T/W):

After Lab:

How it went:

Day 8

Materials required:

Day of Lab (R/F):

After Lab:

How it went:
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