<noautolink> ---+!! Reagent and Buffer Recipes %TOC% ---++ General calculation resources * [[http://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/mass-molarity-calculator.html][Sigma – Mass Molarity Calculator]] * [[http://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/solution-dilution-calculator.html][Sigma – Solution Dilution Calculator]] ---++ 50x TAE Tris•Acetate•EDTA Used as a buffer for [[https://barricklab.org/twiki/bin/view/Lab/ProceduresStandardAgaroseGel][Agarose Gel Electrophoresis]]. | 242 g | Tris base | | 57.1 mL | Glacial acetic acid | | 100 mL | 0.5 M EDTA (pH 8.0) | | to 1 L | ddH<sub>2</sub>0 | Prepare by filling bottle with 700 ml of ddH<sub>2</sub>0 and adding the above chemicals. Adjust volume to 1 L with ddH<sub>2</sub>0. *Unused or left over acetic acid must be disposed of in a chemical waste bottle located in the fume hoods.* To avoid having left over Acetic acid use a serological pipette to measure out the glacial acetic acid. Working concentration is 1x, so measure 400ml of 50x solution in graduated cylinder and then pour into 20 L carboy and fill to 20L with ddH<sub>2</sub>0; if filling a 10 L carboy use 200 ml of stock. ---++ 5x TBE Tris•Borate•EDTA Used as a buffer for [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsPolyacrylamideGelElectrophoresis][Polyacrylamide Gel Electrophoresis]]. | 54 g | Tris base | | 27.5g | Boric Acid | | 20 mL | 0.5 M EDTA (pH 8.0) | | to 1 L | ddH<sub>2</sub>0 | Working concentration is 1x (for standard gels) or sometimes 0.5x (for PFGE). For 1x, measure 100ml of 5x solution into a 1L graduated cylinder and add ddH<sub>2</sub>O up to 500 ml to make it 1x or up to 1000 ml to make it 0.5x. ---++ 20x SB Agarose Gel Buffer (Sodium Borate) An alternative buffer for [[https://barricklab.org/twiki/bin/view/Lab/ProceduresStandardAgaroseGel][Agarose Gel Electrophoresis]]. | 38.2 g | Borax | | 10 g | Boric Acid | | to 1 L | ddH<sub>2</sub>0 | Mix with a stir bar until everything is dissolved and liquid is clear. Pour into one of the 20 L carboys and fill to 20 L with ddH<sub>2</sub>0 to make it 1x. ---++ 6x Bromophenol Blue Gel Loading Buffer | 30 ml | Glycerol | | 0.01 g | Bromophenol Blue | | to 50 ml | ddH<sub>2</sub>0 | ---++ DNA Ladder with Loading Dye | 375 ul | Loading Buffer (above) | | 250 ul | Ladder | | 325 ul | 10x PCR Buffer | | 3 ml | ddH<sub>2</sub>0 | Makes ~4ml. Aliquot to 1.5 ml centrifuge tubes. *To prepare the Invitrogen 1kb+ ladder:* Create a 1:10 dilution of ladder in water, accounting for the addition of your dye of choice (see example below) | 100 ul | Ladder | | 166.6 ul | 6x Loading Buffer (above) | | 733.4 ul | Milipure H2O | ---++ Stock solutions ---+++ Tris-HCl, 1 M | 121 g Tris base in 800 ml ddH<sub>2</sub>0 | | Adjust to pH 8.0 with HCl | | Mix and add ddH<sub>2</sub>0 to 1 L | ---+++ EDTA, 0.5 M (pH 8.0) | Dissolve 186.1 g Na•EDTA•2H<sub>2</sub>0 (EDTA disodium salt dihydrate) in 700 ml ddH<sub>2</sub>0 | | Adjust pH to 8.0 with 10 M NaOH ( It should take ~ 50ml) | | Mix and add ddH<sub>2</sub>0 to 1 L | ---+++ NaOH, 10 M | Dissolve 400g NaOH in 450 ml ddH<sub>2</sub>0. | | Mix and add ddH<sub>2</sub>0 to 1 L | *%RED% *Warning*: Add NaOH pellets slowly! Dissolving is very, very exothermic. %ENDCOLOR% ---+++ Potassium acetate, 5 M | 29.5 ml glacial acetic acid | | KOH pellets to pH 4.8 (several) | | ddH<sub>2</sub>0 to 100 ml | | Store at room temperature | ---+++ NaCl, 1M | Dissolve 58.4 g of NaCl in 800 ml ddH<sub>2</sub>0 | | Mix and add ddH<sub>2</sub>0 to 1 L | ---+++ CaCl<sub>2</sub>, 1 M | Dissolve 110.9 g of CaCl<sub>2</sub> in 800 ml ddH<sub>2</sub>0 | | Mix and add ddH<sub>2</sub>0 to 1 L | | Aliquot into 2 500 ml bottles and autoclave | ---+++ MgSO<sub>4</sub>, 1 M | Dissolve 120.3 g of MgSO<sub>4</sub> in 800 ml ddH<sub>2</sub>0 | | Mix and add ddH<sub>2</sub>0 to 1 L | | Aliquot into 2 500 ml bottles and autoclave | ---+++ RNase A, 5 mg/ml | Dissolve 100 mg of RNase A in 20 ml of 0.05% glacial acetic acid, and transfer to a 50-ml conical tube | | Place the tube in a boiling-water bath for 15 minutes | | Cool the solution, and neutralize by adding 120 μl of 1 M Tris (pH 8.0) | | Distribute 1 ml aliquote into 1.5 ml MFT, and store at -20 C | ---+++ rNTP, 100 mM For in vitro transcription or deoxyribozyme reactions: 1 Dissolve 1 g of desired NTP in 10 ml ddH<sub>2</sub>O: * ATP - Adenosine 5′-triphosphate disodium salt (MW 551.14) * GTP - Guanosine 5′-triphosphate sodium salt hydrate (MW 523.18) 1 pH to 8.0 with 1 M NaOH. It takes approximately 1.0–1.5 ml. 1 Add ddH<sub>2</sub>O to final volume of: * GTP - 18.14 ml * GTP - 19.11 ml 1 Store at –20°C in 1.0–1.5 ml aliquots. ---+++ HEPES•NaOH, 1M, pH 7.0 pH buffer with less temperature dependence than Tris. To make 100 ml: 1 Dissolve 23.83 g HEPES (Free Acid) in 80 ml ddH<sub>2</sub>O. 1 pH to 7.0 with 6 M NaOH. It takes approximately 2.0 ml. 1 Add ddH<sub>2</sub>O to final volume of 100 ml.
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Topic revision: r19 - 2022-03-22 - KateElston