Revision as of 13:45, 30 January 2007

SAGA deletion Module

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

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 (2 YPD, 2 -ura, 2 -trp per lab group for viability assay)
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
need 2x minipreps of pRS416/day.

must wash and reprobe with dendrimers between this lab and next

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

@@ Line 15: / Line 15: @@
 #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
+#need YPD plates, and -ura and a few -trp (2 YPD, 2 -ura, 2 -trp per lab group for viability assay)
 #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
 #need 2x minipreps of pRS416/day.