Make Tris-Cl, pH 8.0
- here we go again ... how come 'error' has to be a part of 'trial and error'?
Attempt #1 = Fail
- used remaining amount of Tris-HCl
- Promega Corp
- Lot # 283523, molecular biology grade
- Cat # H5121, 100g, MW=157.56
- Mixed 18.5g Tris-HCl with approximately 150mL NanoPure H2O
- Heated on hotplate with stirbar until dissolved
- Measured pH using electronic pH meter: pH = 4.12 :(
- added remaining 75mL (to total 234.8mL) and put in storage bottle, labelled "0.5M Tris-Cl, pH 4.12, TKL, 23 November 2010"
- everything I read said to use HCl to bring this mixture to the desired pH (8.0), but it can only go more acidic! How do I get it more basic? Can I use NaOH? Google searches yielded nothing, neither did text messages
Attempt #2
- found another container ... Tris
- Fisher Scientific
- Lot # 086040A
- Cat # BP152-500, 500g, FW=121.14
- Mixed 12.12g Tris with approximately 150mL NanoPure H2O
- Heated on hotplate with stirbar until dissolved
- Measured pH using electronic pH meter; used concentrated aqueous HCl to bring pH to 8.0
- Let sit for 1 hour (went to class)
- pH rose to 8.16, so added HCl to bring back down
- Let sit for another 30 min until stable at pH 8.0
- Added remaining water to bring total volume to 200mL
- Transfered to glass bottle, half-turned screw top (for very loose fit), autoclave tape connecting bottle to cap
- Labelled as "0.5M Tris-Cl, pH 8.0, TKL 23 November 2010"
- Autoclave: seems to have started fine (I waited for a couple minutes until it made clanking sound), but upon return 1 hour later, alarm was sounding
- used: left autoclave, Setting #3 (Liquid, 45 min, 121°C)
- autoclave tape was darkened, so Tris-Cl got sterilized ... considered good!
- re-pHed when cool to confirm ... pH 8.01 ... yay!
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