(r19) Barrick Lab :: ProtocolsReagentRecipes

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---+!! Reagent and Buffer Recipes

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---++ General calculation resources

   * [[http://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/mass-molarity-calculator.html][Sigma &ndash; Mass Molarity Calculator]]
   * [[http://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/solution-dilution-calculator.html][Sigma &ndash; Solution Dilution Calculator]]

---++ 50x TAE Tris&bull;Acetate&bull;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&bull;Borate&bull;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 &mu;l 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&prime;-triphosphate disodium salt (MW 551.14)
      * GTP - Guanosine 5&prime;-triphosphate sodium salt hydrate (MW 523.18)
   1 pH to 8.0 with 1 M NaOH. It takes approximately 1.0&ndash;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 &ndash;20°C in 1.0&ndash;1.5 ml aliquots.

---+++ HEPES&bull;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.
Barrick Lab > ProtocolList > ProtocolsReagentRecipes
Contributors to this topic
JeffreyBarrick, CraigBarnhart, IsaacGifford, AlvaroRodriguez, KateElston, JordanMonk, SimonDAlton, LindseyWolf
Topic revision: r19 - 2022-03-22 - 19:00:11 - Main.KateElston