20.109(S12): TA notes for module 1

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

Day 1: Amplify aptamer-encoding DNA

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.

Day 2: Purify aptamer-encoding DNA

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
• 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:

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 - 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:

Day 4: Purify RNA and run affinity column

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.

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 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:

Day 6: Post-selection IVT and journal club

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

Day 7: Aptamer binding assay

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:

Day 8: Journal club

Materials required:

• None - strictly a journal club day.