20.109(S07): TA's notes for module 3: Difference between revisions
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===Notes:M3D1=== | ===Notes:M3D1=== | ||
#need forward and reverse primers for all non-essential SAGA subunits ordered, validated and available | #need forward and reverse primers for all non-essential SAGA subunits ordered, validated and available | ||
#need template available | #need template (pRS406) available | ||
#need 2.5X PCR "master mix" made up | #need 2.5X PCR "master mix" made up | ||
#have students open Primer Record Handout to use as a guide in primer design | #have students open Primer Record Handout to use as a guide in primer design |
Revision as of 12:48, 30 January 2007
SAGA deletion Module
General notes
Handouts
File:Primer Record Handout Worksheet.doc
Daily Notes
Notes:M3D1
- need forward and reverse primers for all non-essential SAGA subunits ordered, validated and available
- need template (pRS406) available
- need 2.5X PCR "master mix" made up
- have students open Primer Record Handout to use as a guide in primer design
Notes:M3D2
- need quiz for this day to start lab
- need FY2068 ON then subcultured flask growing for lab
- need YPD plates, and -ura and a few -trp
- aliquots for each pair of students: 1 ml ON culture, 1 ml of log phase, bottles of sterile H20 (students can keep these in their drawers if that's easier), cuvettes
- need 2x minipreps of pRS416/day.
Notes:M3D3
Notes:M3D4
Notes:M3D5
must wash and reprobe with dendrimers between this lab and next
Notes:M3D6
Recipes/Reagents
- YPD
- 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
- Zymolyase Stock: MP Biomed #32093 = 100,000U/g so 10 mg/100 ul Y1 gives 100U/10 ul.
- Working spheroplast solution (per sample): 1 ml Y1 +10 ul Zymolylase stock + 1 ul BME.
- RLT+BME for RNA prep; 1 ml RLT from quiagen + 10 ul BME. Use 350 ul/sample
- 0.5M NaOH/0.5M EDTA: 0.1g NaOH to 5 ml 0.5M EDTA (check this calculation).
- 6X SSC/0.005% Triton: 150 ml 20X SSC, 25 ul TritonX-100, 350 ml H20 (how many bottles needed?)
- 2XSSC/0.0016% Triton: dilute 6X 1:3
- 0.2X SSC/0.00016% Triton: dilute 6X 1:30