# User:Tara K. Luckau/Notebook/Team ConGen/2010/11/23

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## 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

• ${\displaystyle 18.5\mathrm {g} \bullet {\tfrac {\mathrm {mol} }{157.56\mathrm {g} }}\ \bullet {\tfrac {\mathrm {L} }{0.5\mathrm {mol} }}\ \bullet {\tfrac {1000\mathrm {mL} }{\mathrm {L} }}\ =234.8\mathrm {mL} }$

1. Mixed 18.5g Tris-HCl with approximately 150mL NanoPure H2O
2. Heated on hotplate with stirbar until dissolved
3. Measured pH using electronic pH meter: pH = 4.12  :(
4. 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

• ${\displaystyle 200\mathrm {mL} \bullet {\tfrac {\mathrm {L} }{1000\mathrm {mL} }}\ \bullet {\tfrac {0.5\mathrm {mol} }{\mathrm {L} }}\ \bullet {\tfrac {121.14\mathrm {g} }{\mathrm {mol} }}\ =12.114\mathrm {g} }$

1. Mixed 12.12g Tris with approximately 150mL NanoPure H2O
2. Heated on hotplate with stirbar until dissolved
3. 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
4. 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"
5. 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!
6. re-pHed when cool to confirm ... pH 8.01 ... yay!