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

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)

Day of Lab

• 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

• 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 detetion kit
• TBS-T

Day of Lab

• Need to write/give/grade quiz

Instructor's Bench

• rack of 50 ml conical tubes
• rack of 15 ml conical tubes
• digital cameras

Student's Benches

Post Lab Prep

• scan or photograph Western data and post

Day 6

Journal club so no prep needed (yay!)

Day 7

Materials required

Day of Lab

• Need to write/give/grade quiz

Instructor's Bench Student's Benches Post Lab Prep

Day 8

Data analysis day so no prep needed (yay!)

Materials

• Culture of FY2068 (genotype: MAT(alpha) ura3-52 his3D200 leu2D1 lys2-128delta)
  • - log phase (dilute in morning) – 1ml/team + 10ml/team
  • - overnight culture –1ml/team

• Plates
  • - YPD (2plates/team)
  • - -trp (2plates/team)
  • - -ura (2plates/team)

Notes:M3D4

Materials

• Sterile toothpicks
• Streak of FY2068 – 1 colony/team
• 2.5X PCR Master mix – 50ul/team
• forward primer (specific)
• reverse primer (URA3) – 2.5ul/team

• YPD (liquid) – 10ml/team
• Sterile tubes – 4/team
• Sterile dowels – 4/team

Notes:M3D5

Materials

• 1% agarose gels (10 lanes) – 1/two teams
• Loading dye – 10ul/team
• 100bp marker – 10ul/two teams
• TAE running buffer
• Film – 1exposure/two teams

• 96-well dish – 1/team
• Plates (have several kinds available for phenotypic testing – 1per kind/team)

**-YPD **-YPG **-YPAc **-YP + 3% form **-SC-lys

• Muti-channel pipette – 1/team (or share)

• Zymolase-supplemented Y1 – 5ml/team
• RNase-away solution
• RNA-dedicated pipetmen
• RNase-free tubes – 5/team
• Rnase-free water – 300ul/team
• Sterile water – 2475ul/team
• Qiagen RNA-easy miniprep kit
  • - RLT+BME solution – 1750ul/team
  • - 70% EtOH – 1750ul/team
  • - spin tubes – 5/team
  • - RW1 solution – 3500ul/team
  • - RPE solution – 2500ul/team

Notes:M3D6

Materials

• Rnase-free tubes – 2/team
• Rnase-free water
• Genisphere cDNA synthesis kit, etc.
  • - Capture sequence I, vial 11, red – 1ul/team
  • - Capture sequence II, vial 11, blue – 1ul/team
  • - CDNA synthesis cocktail – 18ul/team
    • o Superscript first strand buffer
    • o DTT
    • o Superase-In
    • o DNTPs
    • o Superscript II Enzyme
  • - 0.5M NaOH/0.5M EDTA – 7ul/team
  • - 1M Tris, pH7 – 10ul/team
  • - 2X hybridization buffer – 100ul/team
• Yeast v2 DNA microarrays (Agilent) – 1/team

• 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
• Nitrogen gas

must wash and reprobe with dendrimers between this lab and next

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