Standard molecular biology reagents, buffers, and experimental protocols used in the Moniri Laboratory.
1.0 g Ampicillin Add dH2O to 10 ml Sterile filter with 0.2um filter Aliquot ~ 1ml, freeze @ -20°C
2.382 g IPTG Add dH2O to 10 ml Sterile filter with 0.2um filter Aliquot ~ 1ml, freeze @ -20°C
682 mg kanamycin sulfate (Invitrogen stock pwd is 0.734 ug/mg pwd) Add dH2O to 10 ml Sterile filter with 0.2um filter Aliquot ~ 1ml, freeze @ -20°C
use 1mg/ml final In hood, 1.4 g G418 (di)sulfate powder (based on active weight, usually ~700ug active/mg powder) 10 ml sterile dH2O Sterile filter with 0.2um filter into sterile tube Use 1 mg/ml in media for selection 100 ul per 6 ml of media gives ~1.7mg/ml Refresh with new media/G418 every 3 days
16.0 g LB 7.5 g Agar 500 ml dH2O Autoclave, cool Add antibiotic, pour Store at 4°C
80.0 g NaCl (1.37 M final concentration) 2.0 g KCl (26.8 mM) 14.2 g Na2HPO4 or 27.0 g Na2HPO4*7H2O (0.1 M) 2.4 g KH2PO4 (17.6 mM) dH2O to 800 ml stirring in beaker Adjust pH to 7.4 Adjust to 1 L Autoclave
29 g Tris Base 144 g Glycine 10 g SDS or 50 ml of a 20% SDS solution dH2O to 1L Do NOT adjust pH For Non-Denaturing, same as above w/out SDS
25 ml 0.5M Tris-HCL, pH 6.8 20 ml glycerol 20 ml 20% SDS 5 ml 0.1% BPhB dH2O to 100 mL
0.3% → 60–5 kb 0.6% → 20–1 kb 0.7% → 10–0.8 kb 0.9% → 7–0.5 kb 1.2% → 6–0.4 kb 1.5% → 4–0.2 kb 2.0% → 3–0.1 kb
1500 ul bacterial broth 500 ul sterile 60% glycerol Label, freeze at -80°C
30 g Tris Base 88 g NaCl 2 g KCl 970 mL dH2O Bring pH to 7.5 5 ml Tween-20 Adjust to 1 L
30 g Tris Base 88 g NaCl 2 g KCl 970 mL dH2O Bring pH to 7.5 Adjust to 1 L
0.8 ml b-ME 10 ml of 20% SDS 12.5 ml 0.5 M Tris-HCl pH6.8 qs with dH2O to 100 mL Protocol 1) Add membranes and buffer to small plastic box with tight fitting lid 2) Incubate 50°C for 45 minutes 3) Rinse membrane with dH2O 4) Wash 3x in TBST, 5 min each 5) Take on to blocking step
Dissolve 188mg glycine in 75 ml dH2O Adjust pH to 2 with HCl Add 5ml 20% SDS (1% final) QS to 100 ml with dH2O Protocol 1) Warm buffer to 50°C for 15-20 min 2) Soak blot in buffer in container with lid at RT for 30 min 3) Rinse blot with dH2O 4-5 times 4) Wash 3 times with TBST ~5 min each 5) Take on to blocking step
30.25 g Tris Base 144 g Glycine Add 0.1% SDS only if protein of interest >100KDa For 1X, dilute to by: 100 ml 10X Transfer Buffer 200 ml MeOH 700 ml dH2O
5.82 g Tris Base (48mM) 2.93 g Glycine (39mM) Dissolve in 700 ml dH2O Add 200 ml MeOH Add 3.75 ml of 10% SDS (or 1.875 ml of 20% or 0.0375 g SDS) (0.0375%) Adjust to 1 L pH should be ~9.2, do NOT adjust
48.4 g Tris Base 11.42 ml Glacial Acetic Acid 20 ml 0.5 M EDTA (pH 8) dH2O to 1 L
25 mM Tris-HCl (pH 7.6) 150 mM NaCl 1% NP40 1% Na Deoxycholate 0.1% SDS - Add 12.5 ml of 1 M Tris-HCl - Add 15 ml of 5 M NaCl - Add 5 ml of NP40 - Add 50 ml of 10% NaDeoxycholate stock, or add 5 g of NaDeoxycholate powder - Add 2.5 ml of 20% SDS or add 0.5 g of SDS powder - dH2O to 500 ml For non-denaturing (e.g., Co-IP), remove SDS and Na Deoxycholate, replace NP40 above with 0.5% final concentration.
1.5mM MgSO4 4.7mM KCl 1.3 mM CaCl2 1.2mM KH2PO4 25mM NaHCO3 11.7mM Glucose 118mM NaCl pH Adjusted on day of use with O2/CO2 Gas - 369.7mgMgSO4 -350 mg KCl -191mg CaCl2 -163.3mg KH2PO4 -2.1g NaHCO3 -2.108g Glucose -6.895g NaCl -1000ml diH2O
25mM HEPES pH 7.5 1mM EDTA 1% SDS 10µM Neocuproine -Add 3.25g of HEPES -186mg EDTA -1mg Neocuproine -5g SDS -450 ml diH2O -Adjust pH to 7.5 -qs to 500ml For HENS10 dilute HENS buffer 1:10 in diH2O
20 mM HEPES pH 7.5 20mM HEPES pH 7.5 1mM EDTA 100mM NaCl 0.5% Triton -Add 2.6g HEPES -186mg EDTA -2.922g NaCl -450ml diH2O -Adjust pH to 7.5 -Add 2.5 ml Triton-X 100 -qs to 500ml