20.109(S12): TA notes for module 1

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20.109(S12): Laboratory Fundamentals of Biological Engineering

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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

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.
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