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

SAGA deletion Module

Daily Notes

Notes:M3D5

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