20.109(S12): TA notes for module 1
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General notes
S10 and S11 material below; revision for S12 in progress - currently at D4
Scheme:
Each pair of students will prepare aptamer-encoding DNA, then the actual RNA aptamer, and each student will do her/his own column selection. Partners will do the same ratio of 6-5:8-12 and vary column wash number. In S12 a couple of groups may also vary aptamer:bead ratio if desired.
Key preparation:
- Test run: in S11 the 8-12 peak looked off. The bacterial and/or DNA stocks must be tested with the same buffer, etc. to be used during the semester to be certain no element is compromised.
- Chemical stocks to make ahead of time, enough for full run of module (see Google Doc)
- all are done unless otherwise noted due to Agi running pilots this fall
- selection buffer
- 10-15 mM heme
- G7 buffer
- Biological materials to have ready
- assume about 3-4 groups will not have success at any given stage (usually more like 1-2)
- D2 extra 6-5 and 8-12 DNA fragments to be run on extra gel
- check gel at 30 min mark for missing bands, run as many fragments as needed for subsequent 30 min
- possibly run all out quickly on a gel to make sure students have product, and have extra ready for D3 if not
- D4 have 6-5 and 8-12 RNA, especially 6-5
- Reaction recovery notes
- typical recovery on D2 gel is 40-45 ng/uL
- typical IVT yield is 6-10 nmol, with less for 8-12 than 6-5
- RT-PCR test actually showed slight advantage for 6-5 over 8-12, about X % (tk LOOK UP 10ish i think)
- Notes on using Cary spec versus Beckman
- Going 350-425 nm on Beckman in 0.1 nm steps takes ~ 30 sec
- Going 350-800 on Cary in 0.5 nm steps take 40 sec, in 1 nm steps 22 sec
Materials required:
The following items should be in one ice bucket shared per two bench spaces; one group on the 3-group-wide bench gets own bucket (Yellow or Blue), so 4 buckets total. Aliquots should be clearly labeled and ideally color-coded to avoid mix-ups.
- For each item below, distribute enough for 3 rxns + 15% (1 rxn in solo team's bucket)
- PCR Mastermix (from 5')
- Forward and reverse primers
- Original stock is 100 μM, intermediate is 20 μM
- Distribute 20 μM each primer in separate tubes
- DNA plasmid for aptamers 6-5 and 8-12.
- 10.07.11 samples, original stocks are 360 and 245 μg/mL for 6-5, 8-12 respectively
- I have been adding ~ 2.5 ng by doing sequential 1:100 and 1:10 dilutions, then adding 7-10 μL
- For students' case, first dilute the plasmids somewhat more/less than 1:10, to exactly 25 μg/mL each, and let that be their original stock
- Used to require three dilutions due to higher concentration but now they can just do two
Day of Lab (R/F):
- Thaw PCR Mastermix and mix well
- Thaw and aliquot primers, diluted plasmid
- Aliquot water in which to dilute plasmids
After Lab:
- Freeze PCR samples when done.
Materials required:
On teaching bench unless otherwise noted.
- DNA gel
- 2:1 HR agarose gels ready on gel bench
- Ideally made a day in advance, or at least 3 hrs ahead, so they set thoroughly (cover w/plastic wrap to retain moisture)
- Overall 3% gel: 2 parts high resolution to 1 part standard agarose
- For 100 ml of 1X TAE: 2 g high res agarose and 1 g standard agarose
- Make sure to fully dissolve agarose in TAE before microwaving or insoluble gel pieces will form
- Cook thoroughly (until clear), stopping frequently to prevent boilover and gently swirling to mix
- Allow to cool for 1-2 minutes, then add 1 ul sybr safe per 10 ml gel -> 10 ul sybr safe for 100 ml gel
- Do NOT keep the gel in refrigerator overnight
- To make 1X TAE: 20 ul of 50X TAE mixed with 980 ul H2O
- Aliquots of 100 bp ladder (2-3 total) in orange freeze boxes on gel bench
- Aliquots of loading dye - 1 per pair
- for three reactions (6-5, 8-12, control), aliquot 15 ul of XC loading dye
- 2:1 HR agarose gels ready on gel bench
- On gel bench
- Put up signs with sample table for reference.
- Put out nitrile gloves.
- DNA purification
- Qiagen columns and buffers from kit
- Aliquots of isopropanol
- Aliquots of pH 7 water
Day of Lab (T/W):
- Quiz (prepared by TA)
- Thaw PCR products and DNA ladder shortly before lab.
- Remind students to weigh their eppendorfs for the gel slabs ahead of time.
- Turn on water bath so it has plenty of warm-up time, e.g. when gels are started. Kept across from gel bench.
- Test if all students have product after 30 min runtime; if missing product, run teaching aliquots for them on a fresh gel (30 min enough).
- Emphatically warn students about cutting small gel slices.
- Collect and freeze purified DNA at the end of lab.
After Lab
- Turn water bath back off.
How it went:
Materials required:
On teaching bench unless otherwise noted.
- RNase free materials
- Taped off area, bench paper, note about wearing gloves
- Tip boxes, large and small
- RNase away - keep in a box away from sunlight!
- Eppendorf tubes
- IVT
- Aliquots of NTPs, T7, G7 buffer, KOH, pyrophosphatase (see google doc).
- One per every 2 groups in their own ice bucket, enough for 5 rxns.
Day of Lab (R/F):
- Quiz (prepared by TA)
- Thaw their DNA shortly before lab.
- Make sure 37 °C block is on.
After Lab:
- After 4 hrs, RNA samples to -80 °C freezer
How it went:
Materials required:
On teaching bench unless otherwise noted.
- Equilibrate bead slurry in advance with SB.
- Not too far in advance though, as preservative will be diluted out.
- Prepare each eppendorf individually instead of in a larger conical tube — too hard to split up afterward.
- Need 100μL beads per person in an eppendorf tube (total 2 per group).
- Add 200μL hemin slurrry to tube and wash twice with 1X selection buffer.
- Use 1 mL SB, 1 min nutate, 1 min spin at 1000 rcf per wash.
- Distribute beads in total 1mL volume to keep them from sticking to sides of tube.
- One 37 °C heat block, one 70 °C heat block ready.
- Nutators up front. (Plus foil.)
- RNase free materials available as on D3.
- Extra ice bucket up front to collect RNA samples.
- Lots of Selection Buffer!
- Students use approximately 10mL per group (5mL per sample/aptamer-ratio); provide in a conical tubes.
- Figure out in Google Doc.
- Part 1
- DNase aliquotted on ice, 1 per group, approx 20 μL.
- Bring BioSpin columns out just ahead of time, or as needed (keep chilled)-- set on top of ice?
- Part 2
- Clean water, 3 mL per group to be safe.
- 3 cuvettes per group.
- Part 3
- Beads in SB (see above).
- Per group, aliquot 200 μL of 125 μg/mL tRNA (1:100 of stock) and keep on ice.
- Dilution in SB since students will be adding large quantities.
- Part 4
- Ring-stand with two grips per group.
- Poly-prep columns and caps up front (1 per student).
- Make fresh heme dilution to 2.5mM (from 12.5 mM stock, 12.21.11 prep); each student/sample will need 200μL: aliquot ~450-500μL per group.
- Part 5
- Can be aliquotted or at least thawed during lab, not needed for a while. On ice.
- Couple epps with 10-15 μL glycogen each-- to be shared among 2ish groups.
- Few epps ammonium acetate, >=100 μL each.
- One epp per group ethanol, 1.5 mL each.
Day of Lab (R/F):
- Short quiz (prepared by TA)
- Thaw student IVTs at last-minute, along with any reagents to be thawed.
- Help students move through the lab in a timely fashion.
- During the first half-hour, instructor checks their RNA calcs (FNT) and flags any problems while TA helps with column prep.
How it went:
This day tends to run long. Having extra 6-5 and 8-12 at the ready, along with worksheets for checking student calcs, is key.
Materials required:
- HR gel (1 per day)
- Aliquots of ethanol (room temp), 5 mL per group
- Aliquots of 70% ethanol
- Aliquots of RNase-free water, 110 μL per group
- Ice bucket per 2 groups
- Master Mix, 4 rxns + 15-20% per ice bucket (see google doc for recipe)
- PCR tubes
- Loading dye aliquots (minimum 10 μL per group, can share)
Day of Lab (T/W):
- Quiz (prepared by TA)
- When students get to the drying step...
- Put out cold boxes
- Check student samples to make sure they remove enough ethanol!!!
- At the end, run and photograph gel with RT-PCR samples and ladder
How it went:
Materials required:
- As Day 3, but half the amounts of everything.
- S11: Gave the students a Mastermix of IVT materials to speed-up the prep so students could get to Journal Club.
Day of Lab (R/F):
- Quiz (prepared by TA)
- TA runs lab while Agi sets up journal club room
Materials required:
- Part 1
- As Day 4 parts 1 and 2
- Ice bucket per group, lower DNase to 10 uL.
- Part 2
- When use lab rather than Cary spec, have USB hooked up before turning on/before lab
- 2x SB, 1.7 mL per group
- 6 μM heme stock, 1.2 mL per group
- From ~ 12.5 mM stock solution in DMSO in several steps.
- For example, first to ~1 mM in SB , then 100-fold to 10 uM, then to 6 uM. Or in two steps if preferred.
- For original heme stock, dab a bit of solid into the solvent, then measure at 405 nm (extinction coefficient is 180 mM-1 cm-1).
- 7 cuvettes. If DU, 7.5 mm; if Cary, 15 mm height.
Day of Lab (T/W):
- Quiz (prepared by TA)
- Turn on spec and the UV lamp partway through lab
- Again, make sure students dilute RNA in selection buffer and not water!
How it went:
Materials required:
- None - strictly a journal club day.
