1% Blotto 1 liter TTBS (aka TBST) 50 g nonfat dry milk 10 g BSA (Albumin, Bovine) Spike with 2 of 5% Sodium Azide (in hood) Shake it up and put it in the fridge Coomassie Stain 50% MeOH 10% Acetic Acid 0.05% Brilliant Blue R-250 note: dissolve brilliant blue in methanol before adding acetic acid and water For 200 Coomassie Stain: 100 MeOH 20 Acetic Acid 0.1 g Brilliant Blue R-250 80 ml H 2 0 Destain 50% MeOH 40% milli-q water 10% Acetic Acid For 5 L total 2.5 liters Methanol 2 liters milli-q 0.5 liters Acetic Acid Destain (fast) 10% Acetic Acid 90% milli-q water Eppendorph tubes autoclave gravity cycle Glass Pipets autoclave gravity cycle HSE Buffer 20mM Hepes (ph 7.4) 150mM NaCl 5mM EDTA, containing protease inhibitors lastly add 1% Triton 1
IP Buffer 10mM phosphate 150mM NaCl 5mM EDTA 5mM EGTA 1.0% Triton X-100 (if it s without triton, make up volume with water) Prep of phosphate solutions: Solution A: 27.6g NaH2PO4.H2O per liter (0.2M) Solution B: 53.65g Na2HPO4.7H2O per liter (0.2M) Prep of 0.1M Sodium phosphate buffer: 19.0 solution A 81.0 solution B ph should be 7.4 IPTG (make 20 total) 1 molar solution add 238.3g per 1 liter milli-q water 4.77g IPTG / 20 milli-q water store in 4 degrees C Kan (makes 30 total) concentration needs to be 50 mg/ 1.5g Kan/30 make 30, 1 aliquots store in freezer LB broth 25.0 g LB broth per liter of milli-q water mix lightly autoclave (fill tub to about an inch deep in water) autoclave liquid cycle 30 minutes 10X Loading buffer (for DNA gels).021g Bromophenol Blue.021g Xylene Cyanol 4 EDTA (want 0.2M EDTA) 5g glycerol makes about 10 NaF (makes 50 total) 1 molar solution and 41.99 g/liter = 1molar you want 2.0995 g/ 50 2
1X PBS (makes 20 liters total) 160g NaCl 4.0g KCl 23.0 g Na2HPO4 (Sodium Phosphate) 4.0 g KH2PO4 (Potassium Phosphate) thoroughly mix in about 1 L of milli-q water Adjust to ph 7.2 Filter Bring volume to 20 liters with milli-q water Phosphoric Acid (H3PO4) 8.0 / 1 liter milli-q water Protease Inhibitors 100mM AEBSF (-20*C) 0.2397 g AEBSF Bring volume to 10 with water Note that the bottle is stored at RT and the aliquots are at -20 degrees C. 1M Benzamidine 1.566g benzamidine Bring volume to 10 with water note that the bottle is stored at 4 degrees C, and the aliquots are at -20 degrees C Leupeptin/Pepstatin (2 mg/) 2 mg/ Leupeptin and 2 mg/ Pepstatin bring volume to 10 with MeOH Protein gel (stacking gel) Rinse off the gel with water pour stacking gel boil samples for about 5 minutes get running buffer ready (200 running buffer and 800 milli-q water, and mix) put rig together and pour in buffer load gel and run at 150 Chloramphenicol plates stocks: 25 mg/, 1 /1L LB For Bacs final concentration should be 12.5ug/, all other vectors use 25ug/ 3
RIPA buffer (1 liter) 8.76 g NaCl 10 Tris, ph 7.2 (of 1M Tris) 10 of 10% SDS 1 of Triton X-100 10g Deoxylcholate 10 of.5m EDTA recommended: filter 10X Running Buffer (5L) 151.5g TrisBase 720.5g Glycine 50g SDS mix in milli-q water to 5L Lab Recipes 2X Sample Buffer (makes 20 total) 9.5 of milli-q water 2.5 of 0.5 M Tris-HCl ph 6.8 2.0 of Glycerol (same as Glycerin) 4.0 of 10% (w/v) SDS (Sodium Lauryl Sulfate) 1.0 of 2-mercaptoethanol (do this under the hood because this stinks) 1.0 of 1.5% Bromophenol Blue let the solution mix with a stir bar for a while Put 1 of sol n into each 1.5 eppendorph tube and place in freezer. 5X Sample Buffer (makes 20 total) 3.12 of 2M Tris HCl ph 6.8 5 2-Mercaptoethanol (use in hood) 1.88 milli-q water 10 Glycerol 2.0 g SDS (Sodium Lauryl Sulfate) small amount of Bromophenol Blue this makes 20, 1 aliquots SOB Medium (1 Liter) 20g Tryptone 5g Yeast extract 0.584g NaCl 0.186g KCl Dissolve in 1 L dih2o. Adjust ph to 7.0 Autoclave Cool to <50*C. Then add 10 2M Mg2+ Stock 4
SOC (100 ) 99 SOB medium 1 2M Glucose Stock Stacking gel take a 3 aliquot add 15 micro-liters of 10% APS (make sure it s less than a week old) add 5 micro-liters of temed mix it and pour onto gel put comb in and let sit for about 20 minutes Stripping Buffer 15g Glycine 900 H2O 1 Tween-20 adjust ph to 2.5 (use 12N HCl) QS to 1L filter sterilize 1X TAE (makes 20 liters) 96.8 Tris 22.84 Acetic Acid 40.0 0.5M EDTA adjust ph to 8.8 filter 1X TBE (makes 20 L total) 242.4g Trizma Base (same as Tris) 110g Boric Acid 14.8g Na2EDTA (look under EDTA but make sure it has sodium) dissolve in about 1 L adjust to ph 8.3 filter bring volume to 20 liters with milli-q water 10X TBE (makes 1L total) 121.2g Trizma Base (same as Tris) 55g Boric Acid 7.4g Na2EDTA (look under EDTA but make sure it has sodium) adjust to ph 8.3 filter bring volume to 1 liters with milli-q water 5
10X Transfer (makes 2 L total) 60g Tris 288g Glycine bring volume to 2L with milli-q water Lab Recipes 1X TBST (aka TTBS makes 10 L total) 12.2g Tris 87.65g NaCl 5 Tween-20 (0.05% final) mix this up in about 1 L of milli-q water bring up ph to 8.0 bring volume to 10 L 50X TAE (1 L) 242g Tris 57.1 Glacial Acetic Acid 37.2g Na2EDTA 2H2O ph ~ 8.5 Filter 20X NuPAGE MOPS SDS (500 ) 104.6g MOPS 60.6g Tris 10g SDS 3g Na2EDTA2H2O (~ph 7.7) 15% Gels 10 gels ~ 60 13.8 H2O 30.0 30% Acrylamide 15.0 1.5M Tris ph 8.8 0.6 10% SDS 0.6 10% APS 0.06 (60 microl) TEMED 10% Gels- 10 gels ~ 60 23.8 H2O 20.0 30% Acrylamide 15.0 1.5M tris ph 8.8 0.6 10% SDS 0.6 10% APS 0.06 (60 microl) TEMED 6
7.5% Gels- 10 gels ~ 60 28.8 H2O 15.0 30% Acrylamide 15.0 1.5M tris ph 8.8 0.6 10% SDS 0.6 10% APS 0.06 (60 microl) TEMED 4% Gel Solution for Stacking 133 30% Acrylamide solution 250 0.5 M tris, ph 6.8 10 10% SDS 607 H2O Lab Recipes FSGB with sodium azide (200 ml) 5 g BSA 4 ml 1% Na Azide 1g 45% FS Gelatin Dissolve BSA into 150mL 1X PBS Add 1 g FS Gelatin (45%) Add 4mL sodium azide (1% stock solution) (or 0.08g) Bring to final volume of 200 ml with 1X PBS Filter sterilize in hood and aliquot into 20x10mL in 15 ml conical tubes store at -20*C 7