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This page contains general preparation notes for the teaching assistants supporting Module 1 labs. Instructors interested in implementing these labs may contact Agi Stachowiak for additional fine-tuning information.
General Notes
Key Preparation
- Assuming about 3-4 groups don’t have success at any given stage (hopefully more like 1-2), should have extra DNA fragments and RNA aptamers ready in time.
- etc
Scheme: each pair of students will prepare aptamer-encoding DNA, then each one (?) will do an IVT, and each will do her/his own column selection. Partners will do the same ratio of 6-5:8-12 and vary column washes, or perhaps vice-versa. (Maybe latter makes more sense for balanced use of materials?)
Day 1: Amplify Aptamer-Encoding DNA
Materials Required
The following could be up at the teaching bench in one ice bucket total, or could have one ice bucket shared per two bench spaces (4 total).
- PCR Mastermix (from 5')
- Forward and Reverse primers
- Labeled “HindIII SELEX” and “BamHI SELEX”
- Original stock is 100 μM, intermediate is 20 μM
- 3-4 tubes of enough 20 μM primer for 2-3 rxns each + 15% (each tube, not each pair)
- DNA plasmid for aptamers 6-5 and 8-12.
- 06.26.09 samples, original stocks are 2.48 and 2.68 μg/mL for 6-5, 8-12 respectively - ack! this was μg/μL - fixed before students made their samples
- I have been adding ~ 2.5 ng by doing two sequential 1:100 dilutions, then adding 10 μL
- For students’ case, first dilute the plasmids 1:10, and let that be their original stock
- Distribution scheme as for primers above
Day of Lab (R/F)
- Thaw PCR Mastermix and mix well
- Thaw and aliquot primers, diluted plasmid
After Lab
- Freeze PCR samples when done.
Meant for IVT day?
- Prepare PCR Mastermix with T7 polymerase and pyrophosphatase enzymes fresh. Keep on ice.
Day 2: Purify Aptamer-Encoding DNA
Materials Required
On teaching bench unless otherwise noted.
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DNA gel
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2:1 HR agarose gels ready on gel bench
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ideally made a day in advance, or at least 3 hrs ahead, so they set thoroughly (cover w/plastic wrap to retain moisture)
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Aliquots of 100 bp ladder (2-3 total) in orange freeze boxes on gel bench
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Aliquots of loading dye - 1 per pair
- On gel bench
- Put up signs with sample table for reference.
- Put out nitrile gloves.
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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.
- 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).
- Collect and freeze purified DNA at the end of lab.
After Lab
- Turn water bath back off.
How it Went
W/F lab went substantially faster than T/R. Main difference seemed to be telling the students to weigh their eppendorfs for the gel slabs ahead of time, as well as warning about cutting small gel slices even more emphatically.
One group cut out multiple bands per lane, but their purified product looked fine when run on a gel (tested with 5 μL).
Running ~5-10uL of each group’s leftover 6-5 and 8-12 DNA PCR fragments (3% HR Agarose gel + EtBr, 100V for 30 mins) showed that ALL Groups obtained bands of identical size and similar density. Thus, we expect that all groups should have successful IVTs if protocols were followed correctly.
Day 3: Prepare RNA by IVT
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, kept in a box away from sunlight)
- Eppendorf tubes
- IVT
- Aliquots of NTPs, T7, Ppase, KOH (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
All groups on both days finished very quickly (indeed, today is an ideal day for long discussions such as Journal Clubs or writing workshops). Also, the IVT day also allows longer quizzes at the beginning of lab.
Day 4: Purify RNA and Run Affinity Column
Materials Required
On teaching bench unless otherwise noted.
- Equilibrate bead slurry in advance with SB.
- Can do all beads at once, in a big conical tube.
- Not too far in advance though, as preservative will be diluted out.
- e.g. 100μL beads per person in an eppindorf tube (total 2 per group); Give beads in total 1mL volume to keep beads 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 11.1mM stock), 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, check their RNA calcs (hw) and flag any problems.
Day 5: RNA to DNA by RT-PCR
Materials Required
- HR gel (1 per day)
- Aliquots of ethanol (room temp), 5 mL per group
- 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
Day 6: Post-Selection IVT and Journal Club
Materials Required
- As Day 3, but half the amounts of everything.
Day of Lab (R/F)
- Quiz (prepared by TA)
- TA runs lab while Agi sets up journal club room
Day 7: Aptamer Binding Assay
Materials Required
- Part 1
- As for Day 4 parts 1 and 2
- Part 2
- 6 μM heme stock, 1.2 mL per group
- From ~ 1 M stock solution in DMSO
- Dab a bit of solid into the solvent, then measure at 405 nm (extinction coefficient is 180 mM-1 cm-1)
- 2x SB, 1.7 mL per group
Day of Lab (T/W)
- Quiz (prepared by TA)
- Turn on spec and the UV lamp partway through lab
Day 8: Journal Club
Materials Required
- None - strictly a journal club day.
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