(r11) Barrick Lab :: ProtocolsReagentRecipes

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---+!! Reagent and Buffer Recipes

%TOC%

---++50x TAE
| 242 g | Tris base |
| 57.1 ml | Glacial Acetic Acid |
| 18.6 g | EDTA |
| to 1 L | ddH<sub>2</sub>0 |

Prepare by filling bottle with 900 ml of ddH<sub>2</sub>0 and adding the above chemicals.  Adjust volume to 1 L with ddH<sub>2</sub>0.

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.  *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 serological pipettes to measure out the Acetic acid (use 1 50 ml and 1 10 ml).

---++5x TBE Tris•Boric Acid•EDTA 
| 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, so measure 100ml of 5x solution in a 1L graduated cylinder and add ddH<sub>2</sub>O up to 500 ml to make it 1x. Note: Used as a buffer for  [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsPolyacrylamide Gel Electrophoresis][Polyacrylamide Gel Electrophoresis]].

---++ 20x SB Agarose Gel Buffer (Sodium Borate)

| 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. 

---++ 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
| Dissolve 186.1 g NaEDTA 2 dH<sub>2</sub>0 in 700 ml ddH<sub>2</sub>0 |
|Adjust pH to 8.0 with 10 M NaOH (~ 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 |

---+++ 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, 1 M
| Dissolve 110.9 g of CaCl 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 &#956;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 1.0–1.5 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.

---+++ Phusion protein purification
%INCLUDE{ProtocolsReagentsPhusion}%

Included topic: [[ProtocolsReagentsPhusion]]
Barrick Lab > ProtocolList > ProtocolsReagentRecipes
Contributors to this topic
JeffreyBarrick, CraigBarnhart, IsaacGifford, AlvaroRodriguez, LindseyWolf, SimonDAlton, KateElston, JordanMonk
Topic revision: r11 - 2014-04-11 - 17:06:13 - Main.CraigBarnhart