20.109(F08): TA's notes for module 2

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Protein Engineering Module

Current: 20.109(F08)
this is a new module but has techniques that are similar to 20.109(S07) (archive)
and 20.109(F07) (archive)

General notes

Before the term begins:

  1. Grow NB318 in LB+A, 37° overnight and Qiagen prep (one prep/lab period). Use this plasmid (pBS1479) for Day 1 PCR, 1 ul/rxn
  2. Grow NB133 in LB+A, 37° overnight and Qiagen prep (two preps/lab period). Use this plasmid (pRS414) for Day 2 txn, 10 ul/txn
  3. Streak NY411, NY412, NY413 on YPD, 30°
  4. Order from IDT TAP tagging primers, + universal checking primer (NO225), +SPT3 and SPT7 control primers
  5. Order microarrays from Agilent. These are not "in stock" items and they need ~3weeks to print and QC
    • Agilent part number G2519F, Design number 015072
    • also order gaskets for arrays, Agilent part number G2534-60011
  6. In case student's materials don't work, pre-run all PCRs from DAY 1 and verify 1.5 kb band on gel

Daily Notes

Day 1

Materials required:

  1. PCR Master Mix (2.5X), (need ~50 μL per group; make two aliquots large enough for whole class to use)
  2. DI water (one 100 μL aliquot per group)
  3. primers (100 pmol/ul)
  4. template (pBS1479 Q prep'd from NB318, make two aliquots with enough volume for whole class to use)
  5. 2 PCR tubes per group

Day of Lab:

  • No TA quiz needed today since first day of module

Instructor's Bench: (not needed until near the end of lab)

  • Ice bucket
  • PCR Master Mix (thawed on ice only just before first group needs it at end of lab)
  • Sterile H20, (100 ul aliquot/group)
  • Primers for TAP-tagging (thaw only as primers requested)
  • Template for PCR= pBS1479 (need 2 ul/rxn; make two aliquots with large enough volume for whole class to use)
  • Beaker with PCR tubes
  • PCR chill racks from -20° (1 per group)

PCR:
Check the PCR machine has proper protocol called "TAP":

  • Step 1: 94° 5 minutes
  • Step 2: 94° 30 seconds
  • Step 3: 48° 30 seconds
  • Step 4: 72° 4 minutes
    • Repeat steps 2-4 34 more times
  • Step 5: 72C 10min
  • Step 6: 4C hold forever
    • Remember to freeze PCR products when they are ready.

Post Lab Prep

  • The night before Day 2, grow NY411 in 2.5 ml YPD. One tube/two groups.
  • The morning of Day 2, subculture NY411. 2 ml into 20 ml YPD in 125 ml flask RT shaker, at least 4 hours. Prepare one flask/two groups.

Day 2

Materials required

  • PCR purif kit, two columns/group
  • Q-biogene Transformation kit, one kit/two labs is plenty
  • pRS414 positive transformation control DNA, miniprep 4 overnight cultures of NB133 and pool.
  • SC-trp plates
  • 1% agarose gel (1X TAE)

2 days before lab
Grow 3 ml overnight culture of NY411 in YPD at 30° on roller

  • Need one overnight for every two groups
  • Can set up overnights for both days of lab and then leave the cultures at RT needed. Cells will settle but can be vortexed before subculturing on day of lab.

Day of Lab

  • at least 6 hours before lab begins:
    • innoculate 2 ml of overnight culture into 20 ml YPD in 125 ml flask and grow in shaker at room temp
    • no need to put water in shaker
    • OD of cells ready for transformation is ~0.1 or 0.2 when reading a 1:10 dilution at 600 nm.
  • Need to write/give/grade quiz

Instructor's Bench

  • Q-biogene Transformation kit, does not need to be aliquoted but confirm sufficient volumes available
    • - wash solution – will need 3ml/team
    • - competent solution – will need 150 ul/team
    • - transformation solution – 1ml/team
  • transformation + control = pRS414 DNA miniprep – will need 10ul/team, does not need to be aliquoted but confirm sufficient volumes available, make two aliquots with large enough volume for whole class to use
  • Spreaders, EtOH beakers and alcohol burners

Student's Benches

  • SC-trp plates – 4/team

Post Lab Prep

  • Run student purified samples on 1% agarose gel and photograph and post image to wiki.
  • Remove petri dishes from 30° incubator after 3 days and move to 4° until Day 3

Day 3

Materials required

  • YPD plate with single colonies of NY411 and NY412 and NY413 (one plate of each/lab period)
  • sterile toothpicks
  • sterile water
  • PCR tubes
  • Universal reverse TAP primer = NO225, TCA GGT TGA CTT CCC CGC GGA ATT CGC GTC TAC
  • Other TAP-tagging primers as requested by students
  • Positive control primer
    • NO258 for SPT3-TAP strain NY412, TTT AAA GGT GGT AGA CTC AGT TCT AAA CCA ATT ATC ATG tcc atg gaa aag aga aga tg
    • NO259 for SPT7-TAP strain NY413, AAT AGT TCA TTT AGC TTG AGC CTT CCT CGC CTT AAT CAA tcc atg gaa aag aga aga tg
  • PCR master mix (2.5X)
  • YPD for overnights (15 ml aliquot/group)
  • sterile tubes
  • sterile dowels
  • 1% agarose gel (1X TAE), 5 lanes needed/group (four samples, as well as one lane for 100 bp or 1kb markers)

Day of Lab

  • Need to write/give/grade quiz

Instructor's Bench

  • rack of sterile tubes
  • aliquots of YPD
  • sterile toothpicks
  • PCR tubes
  • Ice bucket
  • PCR mastermix ~1 hr into lab
  • PCR chiller racks ~ 1 hr into lab

Student's Benches

  • return student's petri dishes to each group before lab begins

PCR program modification of program called "NK1"

  • Step 1: 95C 4min
  • Step 2: 95C 1 min
  • Step 3: 52C 1 min
  • Step 4: 72C 2 min
    • Repeat steps 2-4 34 more times
  • Step 5: 72C 10min
  • Step 6: 4C hold forever
    • Remember to freeze PCR products when they are ready or run on gel directly. No need to save unless there's a question.

Post Lab Prep

  • Run 5 ul of PCR samples on 1% agarose gel for students, photograph, post image to wiki. Each group will have 4 samples to run on the gel and each group should have a lane with molecular weigh markers. TAP tag should give product of ~554 bp.
  • Remove liquid overnight cultures after 24 hours at 30° and store at 4° until Day 4

Day 4

Materials required

  • box of plastic cuvettes
  • water for diluting cells
  • 1X Sample Buffer with BME
    • 5ml/day should be plenty
    • leave in 1 ml aliquots in hood
  • glass beads for lysis
  • boiling water bath in hood with lid locks for samples
  • Pre-cast gels from BioRad
    • 4-15% gradient gels, 50 ul/well, 10 wells
    • BioRad cat # 161-1158
    • need one gel/two groups
    • can fit two gels/gel box
  • TGS, 10X stock
    • need 1 liter/gel box (i.e. for 4 groups)
  • Kaleidoscope Marker
    • need to boil 25 ul aliquots/protein gel when students are boiling their samples
  • Transfer Buffer for blotting
    • need 1 liter/gel box (i.e. for 4 groups)
  • TBS-T + 5% milk
  • 96 well dish, 1/group
  • YPD plates, SD-his, SD-trp, SD-lys
    • make sure these are stripped according to code that's listed on Day 4 lab protocol
  • 37° and 30° incubators

Day of Lab

  • Need to write/give/grade quiz

Instructor's Bench

  • box of cuvettes
  • glass beads for cell lysis and small measuring eppendorfs
  • Whatman paper for blotting
  • Nitrocellulose
  • Square tupperware dishes
  • stacks of petri plates (YPD, SC-trp, SC-lys, SC-his)
  • multichannel pipettors
  • 1X TBS-T with 5% milk
    • 25 ml/group
  • tupperware for blotting overnight

Student's Benches

  • ice bucket
  • 4 yeast cultures that were grown for each group
  • 50 ml conical with 20 ml sterile water
  • one 96 well dish

Gel running bench

  • protein gel boxes (one box/4 groups)
    • Kaleidoscope Marker
    • need to boil 25 ul aliquots/protein gel when students are boiling their samples
  • 1XTGS (need 1 liter/gel box)
  • Transfer Buffer, keep @ 4° until blot set up (need 1 liter/gel box)

Chemical Fume Hood

  • 1X sample buffer with BME
    • 5 ml should be enough for each lab, divided into several eppendorf tubes
  • Boiling water bath
    • Turn on once protein preps are underway
    • Don't let water boil dry
    • Turn off when all students done

Post Lab Prep

  • put student's yeast cultures back to 4° until Day 5
  • move to 4° any petri plates from 30° or 37° as they grow fully or get contaminated

Day 5

Materials required

  • primary antibody (anti-protein A from Sigma)
  • secondary antibody
  • AP detection kit
  • TBS-T
  • Bench paper
  • Ambion "RiboPure Yeast RNA isolation kit"
    • each kit has enough reagents for 50 purifications
    • one new kit and the leftovers of another should be enough material for 2 lab sections
    • purchased from Applied Biosystems catalog number AM1926
    • use with vortex adapter head from Applied Biosystems, catalog number AM10024
  • Student's lab coats
  • 100% EtOH
    • 5 ml/group in RNase-free Falcon
    • may want to start new bottle of EtOH for this
  • quartz cuvettes

Day of Lab

  • Need to write/give/grade quiz

Instructor's Bench
Place bench paper on instructor's bench and mark "RNA only! Use Gloves!" to leave all RNA purif materials (bolded) there.

  • rack of 50 ml conical tubes
  • rack of 15 ml conical tubes
  • digital cameras
  • ice bucket with primary and secondary antibodies thawed just before class
  • 1X AP developing solution
    • dilute 1 ml of 25X stock into ~24 ml dH2O (one falcon tube/group)
    • can leave developing reagents A and B in freezer until first group needs them
  • 100% EtOH (5 ml/group in RNase-free Falcon)
  • Ambion RiboPure Yeast RNA isolation kit
    • aliquot Elution Solution (125 ul/group)
    • place in 95° heat block on instructor's bench near end of lab
  • 15 ml conicals with 5 ml sterile H2O (one per group)

Student's Benches

Chemical Fume Hood

  • two vortexes should be moved into chemical hood, one with multi-lyse adapter
  • confirm the PCI waste bottle has room enough for new materials

Post Lab Prep

  • scan or photograph Western data and post

Day 6

Journal club so no prep needed (yay!)

Day 7

Materials required

  • Heat blocks for 80°, 65°, and 42°
  • Bench paper
  • Genisphere cDNA synthesis kit, etc.
    • Capture sequence I, vial 11, red – 1ul/team
    • Capture sequence II, vial 11, blue – 1ul/team
    • CDNA synthesis cocktail
      • Superscript first strand buffer
      • DTT
      • Superase-In
      • DNTPs
      • Superscript II Enzyme
  • 0.5M NaOH/50mM EDTA
  • 1M Tris, pH7
  • 2X hybridization buffer
  • Yeast v2 DNA microarrays (Agilent) – 1array/4 groups
  • 20X SSC (Ambion; 3M NaCl, 0.3M NaCitrate)
  • 6xSSC/0.005% Triton X-100
  • 2X SSC/0.0016% Triton X-100
  • 0.2X SSC/0.00016% Triton X-100

Day of Lab

  • Need to write/give/grade quiz

Instructor's Bench

  • thaw RNA just before lab
  • ice bucket
  • cDNA synthesis cocktail just before needed
  • 2 heat blocks, one for 80° and 65° and other for 42°

Student's Benches

Post Lab Prep

  • must wash and reprobe with dendrimers between this lab and next

Day 8

Data analysis day so no prep needed (yay!)

Recipes/Reagents

  1. YPD
    • 10g yeast extract
    • 20g peptone
    • 20g glucose
    • 20g agar (for liquid media, leave agar out!)
    • add 1L water, autoclave 20min, stir to cool
  2. SC-lys, his or trp
  3. 1X Sample Buffer + BME
    • 2.5 ml 2X Sample Buffer
    • 2 ml dH2O
    • 0.5 ml BME, added in hood
  4. 1XTGS
  5. Transfer buffer
    • Trizma base
    • Glycine
    • dissolve in 800 ml dH2O
    • add 200 ml Methanol and store in bottles 4° until needed for blotting
    • in a pinch can re-use the buffer once
  6. TBS-T
    • dilute 10X TBS, 100 ml to 1L with dH2O
    • add 10 ml of 10% Tween (stored in 4° delicase) for final conc of 0.1%
  7. TBS-T + 5% milk
    • add 25 g powdered milk to 500 ml of TBS-T
    • stir plate to dissolve
    • aliquot 25 ml/group
    • store a night or two at 4° if making in advance of day of lab
  8. 0.5M NaOH/0.5M EDTA: 0.1g NaOH, 0.5 ml of 0.5 M EDTA, 4.5 ml H2O
  9. 6X SSC/0.005% Triton: 150 ml 20X SSC, 25 ul TritonX-100, 350 ml H20 (how many bottles needed?)
  10. 2XSSC/0.0016% Triton: dilute 6X 1:3
  11. 0.2X SSC/0.00016% Triton: dilute 6X 1:30
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