20.109(S13): TA notes for module 1
Coming soon!
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:
