20.109(S14): TA notes for module 1: Difference between revisions
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*Place an eppendorf tube rack in the 56 °C oven to warm it up. | *Place an eppendorf tube rack in the 56 °C oven to warm it up. | ||
*Set up the heat block at the front bench. Fill tube slots with water and set at 70 °C. Ensure the heating block can accommodate 2 mL tubes. | *Set up the heat block at the front bench. Fill tube slots with water and set at 70 °C. Ensure the heating block can accommodate 2 mL tubes. | ||
*Arrange the groups that take their samples out of the 56 ° at the same time to spin their samples together. | |||
*Either set up second heat block at 56 °C or turn on the small oven (may be easiest). -Agi will do | *Either set up second heat block at 56 °C or turn on the small oven (may be easiest). -Agi will do | ||
*If the fumehood gets crowded at the end of the lab, while the students are disposing of their Qiagen flow-throughs, you can give the students 50 mL falcon tubes and a spray bottle to dispose the waste. Then dump into the appropriate Qiagen waste container. | *If the fumehood gets crowded at the end of the lab, while the students are disposing of their Qiagen flow-throughs, you can give the students 50 mL falcon tubes and a spray bottle to dispose the waste. Then dump into the appropriate Qiagen waste container. | ||
'''After Lab:''' | '''After Lab:''' | ||
Revision as of 11:17, 7 February 2014
General notes
These TA notes need a fair bit of revision/fleshing out… in progress. I also need to figure out the quiz dates.
See also GoogleDoc for aliquoting amounts.
Scheme: Class-wide, students will test 8-10 fecal samples in replicate, in order to get enough sequencing data for establishing phylogenetic trees. Last year, the two partners in each team prepared different samples, but that created some workflow issues later in the module; this year we will have them prepare duplicates of the same sample.
Key preparation:
- Pre-run entire bird gut microbiota experiment with 1-2 fecal samples, to become familiar with each experiment.
- Pre-test DNA extraction from all 8-10 samples (just through PCR and gel test). During S13 one sample consistently failed.
- Try the primer design day (microsporidia experiment) to be able to answer questions about that experiment also.
- 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:
- Before aliquoting out the cloacal samples, thaw the samples for ~4h at 4°C, and then vortex each cryovial for 2 min.
- 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.
- Place chitinase aliquots on ice.
- 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).
- Place bags of fresh 2 mL tubes, 1.7 mL tubes, Qiagen spin columns, tablets, and column tubes at the front.
- Place Part 1 aliquoted reagents on the front bench. Leave the Part 2 aliquots at the TA bench for now.
- Check ASL and AL for precipitates; pre-heat at about 40-50 °C in the water bath at the TA bench
- Place an eppendorf tube rack in the 56 °C oven to warm it up.
- Set up the heat block at the front bench. Fill tube slots with water and set at 70 °C. Ensure the heating block can accommodate 2 mL tubes.
- Arrange the groups that take their samples out of the 56 ° at the same time to spin their samples together.
- Either set up second heat block at 56 °C or turn on the small oven (may be easiest). -Agi will do
- If the fumehood gets crowded at the end of the lab, while the students are disposing of their Qiagen flow-throughs, you can give the students 50 mL falcon tubes and a spray bottle to dispose the waste. Then dump into the appropriate Qiagen waste container.
After Lab:
- Store extraction samples at -20 °C until next time
How it went:
Day 2
Materials required:
- None :)
Day of Lab (T/W):
How it went:
Day 3
Materials required:
- Their D2 extractions
- PCR components aliquoted (except polymerase) - per two teams sharing, 6 rxns + 30% per component should be reasonable
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
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
Day of Lab (R/F):
- Help prepare journal club room (16-336) in advance.
- Feel free to participate in Q&A sessions of the talks.
Day 7
Materials required:
Day of Lab (T/W):
After Lab:
How it went:
Day 8
Day of Lab (R/F):
- Help prepare journal club room (16-336) in advance.
- Feel free to participate in Q&A sessions of the talks.
