20.109(S07): TA's notes for module 3

SAGA deletion Module

Handouts

File:Primer Record Handout Worksheet.doc

Daily Notes

Notes:M3D1

1. need forward and reverse primers for all non-essential SAGA subunits ordered, validated and available
2. need template (pRS406) available
3. need 2.5X PCR "master mix" made up
4. have students open Primer Record Handout to use as a guide in primer design

Materials

• Forward primer
• Reverse primer
• Template – 40ul/team
• Pcr master mix – 1ul/team

Notes:M3D2

1. need quiz for this day to start lab
2. need FY2068 ON then subcultured flask growing for lab
3. need YPD plates, and -ura and a few -trp (2 YPD, 2 -ura, 2 -trp per lab group for viability assay)
4. aliquots for each pair of students: 1 ml ON culture, 1 ml of log phase for counting and viability assays, additional 10 ml aliquot of log phase for prep of competent cells, bottles of sterile H20 (students can keep these in their drawers if that's easier), cuvettes
5. need 2x minipreps of pRS416/day.

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)

• Q-biogene Transformation kit
  • - wash solution – 3ml/team
  • - competent solution – 50ul/team
  • - pRS416 DNA – 5ul(50 ng)/team
  • - transformation solution – 1ml/team

• SC-ura plates – 4/team

Notes:M3D3

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:M3D4

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:M3D5

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
2. Buffer Y1 for RNA isolation: 1M sorbitol (9.1g/50ml), 0.1M EDTA (10 ml of 0.5M/50 ml), Filter sterilize, leave at RT
3. Zymolyase Stock: MP Biomed #32093 = 100,000U/g so 10 mg/100 ul Y1 gives 100U/10 ul.
4. Working spheroplast solution (per sample): 1 ml Y1 +10 ul Zymolylase stock + 1 ul BME.
5. RLT+BME for RNA prep; 1 ml RLT from quiagen + 10 ul BME. Use 350 ul/sample
6. 0.5M NaOH/0.5M EDTA: 0.1g NaOH to 5 ml 0.5M EDTA (check this calculation).
7. 6X SSC/0.005% Triton: 150 ml 20X SSC, 25 ul TritonX-100, 350 ml H20 (how many bottles needed?)
8. 2XSSC/0.0016% Triton: dilute 6X 1:3
9. 0.2X SSC/0.00016% Triton: dilute 6X 1:30