Solutions for Txn Lab FIR 2007
Solution HEGD (hypotonic buffer) (make FRESH) NUCLEAR EXTRACT ISOLATION Stock soln concentration Vol (ul) for 10 ml Final 1M HEPES pH 7.9 250 1M DTT 10 0.5M EDTA pH 8.0 30 50 % glycerol 2000 *50X complete protease inhibitor mix 200 H2O 7510 Vol (ul) for 2ml Final 25 mM 1M HEPES pH 7.9 50 1 mM 1M DTT 2 1.5mM 0.5M EDTA pH 8.0 6 10% 50% glycerol 400 0.5M 1.0M KCl 1000 1X *50X complete protease inhibitor mix 40 H2O 502 For 0.5 ml Final 100mM 8-Bromo- 100mM 8-Bromo-cAMP Disolve 17.20 mg 8-bromoFinal Conc 25 mM 1 mM 1.5mM 10% 1X
HEGDK (extraction buffer) (make FRESH)
8-Bromo-cAMP Stock Soln. Protien Assay Solutions
2X CREB EMSA Buffer
10mM Tris (7.4) 50mM NaCl 1mM MgCl2 0.5mM EDTA 5% Glycerol 0.5mM DTT 125ug/ml BSA 50 ug/ml dIdC H2O
1M Tris (7.4) 5M NaCl 1M MgCl2 0.5M EDTA 50% Glycerol 1M DTT 20mg/ml BSA 0.5mg/ml
Vol for 1ml 2X Buffer 20 20 2 2 200 1 12.5 200 462.5
Solutions for Txn Lab FIR 2007
ChIP Lysis Buffer 10mM 50mM 1% 1X 0.5M EDTA 1M Tris pH 8.1 10% SDS *50X complete protease inhibitor mix H2O 1M Tris pH 8.1 5M NaCl 0.5M EDTA 10% Triton X-100 10% SDS *50X complete protease inhibitor mix H2O 1M Tris pH 8.1 5M NaCl 0.5M EDTA 10%Triton X-100 10% SDS *50X complete protease inhibitor mix H2O ChIP High-Salt Wash Buffer 20mM 500mM 2mM 1% 0.10% 1X 1M Tris pH 8.1 5M NaCl 0.5M EDTA 10% Triton X-100 10% SDS *50X complete protease inhibitor mix H2O 5M LiCl 10% NP-40 10% Na Deoxycholate 1M Tris pH 8.1 0.5M EDTA *50X complete protease inhibitor mix H2O 1M Tris (8.0) 0.5M EDTA *50X complete protease inhibitor mix H2O 1M NaHCO3 10% SDS H2O
H2O
ChIP Dilution Buffer
16.7mM 167mM 1.22mM 1.10% 0.01% 1X
ChIP Low-Salt Wash Buffer
20mM 150mM 2mM 1% 0.10% 1X
Vol (ul) for 1ml Final 20 50 100 20 790 Vol (ul) for 4ml Final 67 134 9.8 440 4 80 3266 Vol (ul) for 4ml Final 80 120 16 400 40 80 3264 Vol (ul) for 4ml Final 80 400 16 400 40 80 2984 Vol (ul) for 4ml Final 200 400 400 40 8 80 2872 Vol (ul) for 8ml Final 80 16 160 7744 Vol (ul) for 2ml Final 200 200 1600 Vol/rxn
14 2.5 1 0.25 1 1 0.25 5
ChIP LiCl Wash Buffer
250mM 1% 1% 10mM 1mM 1X
ChIP TE
10mM Tris (8.0) 1mM EDTA 1X
ChIP Elution Buffer
100mM 1%
PCR Cocktail 1X 20 mM MgCl2 0.25 mM dNTPs 0.25 uM 0.25 uM
10X PCR Buffer 50 mM MgCl2 25 mM dNTP's *For [6.25 pmol/ul] *Rev [6.25 pmol/ul] Platinum Taq DNA Polymerase DNA Template
Roche protease inhibitor stock
50X Stock Soln. 50X
For 0.2 ml Final Disolve 1 complete mini tablet into 200 ul sterile H2O
* Protease inhibitors must be added fresh to solutions. If you have extra protease inhibitor, store it at and use it for solutions made at a later time.
Solutions for Txn Lab FIR 2007
Solutions for Txn Lab FIR 2007
For 1 ml Final Disolve 43.01 mg 8-
Solutions for Txn Lab FIR 2007
Vol (ul) for 4ml Final 80 200 400 80 3160 Vol (ul) for 15ml Final 251 502 37 1.65 ml 15 300 12.24 ml Vol (ul) for 10ml Final 200 300 40 1 ml 100 200 8.16 ml Vol (ul) for 10ml Final 200 1 ml 40 1 ml 100 200 7.46 ml Vol (ul) for 10ml Final 500 1 ml 1 ml 100 20 200 7.18 ml Vol (ul) for Xml Final
Vol (ul) for Xml Final
Vol/15 rxn
210 37.5 15 3.75 15 15 3.75
For 1 ml Final Dissolve 1 complete or 5 complete mini tablets into 1ml sterile H2O
bitor, store it at -20oC
Solutions for Txn Lab FIR 2007
Reaction Blank 1 2 3 4 5 6 7 8 9 10 11
Protein Sample None BSA BSA BSA BSA BSA BSA BSA BSA MA-10 NE MA-10 NE MA-10 NE
Concentration (ug/ul) 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ? ? ?
Sample Vol. (ul) 0 1 2 3 4 5 6 10 15 1 2 3
Assay Conc. (ug/ml) 0 2 4 6 8 10 12 20 30 ? ? ?
H20 Vol. (ul) 800 799 798 797 796 795 794 790 785 799 798 797
Dye Conc. Vol. (ul) 200 200 200 200 200 200 200 200 200 200 200 200