20.109(S13): TA notes for module 1

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(New page: {{Template:20.109(S13)}} <div style="padding: 10px; width: 640px; border: 5px solid #99FF66;"> Coming soon! ==General notes== '''Scheme:''' '''Key preparation:''' ===Day 1=== '''Ma...)
Current revision (10:47, 10 June 2013) (view source)
(Day 5)
 
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==General notes==
==General notes==
 +
 +
See also GoogleDoc for aliquoting amounts.
'''Scheme:'''  
'''Scheme:'''  
'''Key preparation:'''
'''Key preparation:'''
 +
 +
==Day-by-day==
===Day 1===
===Day 1===
 +
 +
'''Materials required:'''
 +
 +
'''Day of Lab (R/F):'''
 +
 +
'''After Lab:'''
 +
 +
'''How it went:''' Pre-lab ran longer than it should have, leaving students with just over two hours to complete their designs -- not enough time for most.
 +
 +
===Day 2===
 +
 +
'''Materials required:'''
 +
*Bird samples! At 100 &mu;L each in a '''2 mL''' tube
 +
**Use filter pipet tip to transfer and store at -80 &deg;C -- consult Agi about which samples to prepare
 +
*Aliquots of components from QiaAMP stool kit (see GoogleDoc)
 +
**Do NOT put out ethanol until after everyone is at the incubation step; we don't want them to accidentally add it too early
 +
**Some aliquots may be approximate (use your judgment). For example, it will be easier to pipet approximately 0.5 mL into each tube using a serological pipet than to pipet exactly 0.46 mL eight times per day, times several reagents!
 +
**ASL and AL must be aliquoted the day of lab (see below)
 +
*InhibiTEX tablets should be singly available -- pre-cut around them; otherwise it is too easy to push out two instead of one
 +
 +
'''Day of Lab (R/F):'''
 +
*Thaw that day's 100 &mu;L samples in the fridge starting just before lecture. -Agi will do
 +
*Place 4 ice buckets around the room (between pairs) for keeping samples chilled at first.
 +
*Before lab, move samples to the ice bucket nearest the pair that will be using those particular samples (see table on Talk page, ready shortly).
 +
*Check ASL and AL for precipitates; pre-heat at about 40-50 &deg;C in the water bath at the TA bench
 +
*Set up the heat block at the front bench. Fill tube slots with water and set at 70 &deg;C
 +
*Either set up second heat block at 56 &deg;C or turn on the small oven (may be easiest). -Agi will do
 +
 +
'''After Lab:'''
 +
*Store extraction samples at -20 &deg;C until next time
 +
 +
'''How it went:'''
 +
 +
===Day 3===
 +
 +
'''Materials required:'''
 +
*Their D2 extractions
 +
*PCR components aliquoted (except polymerase, see below) - 6 rxns + 30% per component should be reasonable, shared by two teams
 +
 +
'''Day of Lab (R/F):'''
 +
*Agi will take care of polymerase aliquots: 1-2 big aliquots to share, and students will come up to the front bench when they are ready and use the smaller pipet tips for this step
 +
 +
'''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 (R/F):'''
 +
 +
'''After Lab:'''
 +
 +
'''How it went:''' There is an issue with the reaction that causes many clones to fail -- not just to be lacking insert, but to be lacking a chunk of the original vector! (Either F or R priming site becomes missing.) Also, sample #709 produced few colonies and no successful clones. I prepared 10 additional minipreps (6 from student ST, 4 fresh), and all failed. Spot-checked 4 preps, and most ratios were between 1.7-1.9 (one was 1.5) and most A260's were around 0.1 diluted.
 +
 +
===Day 6===
 +
 +
'''Materials required:'''
 +
 +
'''Day of Lab (R/F):'''
 +
 +
'''After Lab:'''
 +
 +
'''How it went:'''
 +
 +
===Day 7===
 +
 +
'''Materials required:'''
 +
 +
'''Day of Lab (R/F):'''
 +
 +
'''After Lab:'''
 +
 +
'''How it went:'''
 +
 +
===Day 8===
'''Materials required:'''  
'''Materials required:'''  

Current revision

20.109(S13): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2013        Assignments       
DNA Engineering        Protein Engineering        Cell Engineering              

Coming soon!

Contents

General notes

See also GoogleDoc for aliquoting amounts.

Scheme:

Key preparation:

Day-by-day

Day 1

Materials required:

Day of Lab (R/F):

After Lab:

How it went: Pre-lab ran longer than it should have, leaving students with just over two hours to complete their designs -- not enough time for most.

Day 2

Materials required:

  • Bird samples! At 100 μL each in a 2 mL tube
    • Use filter pipet tip to transfer and store at -80 °C -- consult Agi about which samples to prepare
  • Aliquots of components from QiaAMP stool kit (see GoogleDoc)
    • Do NOT put out ethanol until after everyone is at the incubation step; we don't want them to accidentally add it too early
    • Some aliquots may be approximate (use your judgment). For example, it will be easier to pipet approximately 0.5 mL into each tube using a serological pipet than to pipet exactly 0.46 mL eight times per day, times several reagents!
    • ASL and AL must be aliquoted the day of lab (see below)
  • InhibiTEX tablets should be singly available -- pre-cut around them; otherwise it is too easy to push out two instead of one

Day of Lab (R/F):

  • Thaw that day's 100 μL samples in the fridge starting just before lecture. -Agi will do
  • Place 4 ice buckets around the room (between pairs) for keeping samples chilled at first.
  • Before lab, move samples to the ice bucket nearest the pair that will be using those particular samples (see table on Talk page, ready shortly).
  • Check ASL and AL for precipitates; pre-heat at about 40-50 °C in the water bath at the TA bench
  • Set up the heat block at the front bench. Fill tube slots with water and set at 70 °C
  • Either set up second heat block at 56 °C or turn on the small oven (may be easiest). -Agi will do

After Lab:

  • Store extraction samples at -20 °C until next time

How it went:

Day 3

Materials required:

  • Their D2 extractions
  • PCR components aliquoted (except polymerase, see below) - 6 rxns + 30% per component should be reasonable, shared by two teams

Day of Lab (R/F):

  • Agi will take care of polymerase aliquots: 1-2 big aliquots to share, and students will come up to the front bench when they are ready and use the smaller pipet tips for this step

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 (R/F):

After Lab:

How it went: There is an issue with the reaction that causes many clones to fail -- not just to be lacking insert, but to be lacking a chunk of the original vector! (Either F or R priming site becomes missing.) Also, sample #709 produced few colonies and no successful clones. I prepared 10 additional minipreps (6 from student ST, 4 fresh), and all failed. Spot-checked 4 preps, and most ratios were between 1.7-1.9 (one was 1.5) and most A260's were around 0.1 diluted.

Day 6

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 7

Materials required:

Day of Lab (R/F):

After Lab:

How it went:

Day 8

Materials required:

Day of Lab (R/F):

After Lab:

How it went:
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