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]]

---++ Gel Electrophoresis

---+++ 50&times; TAE (Tris&bull;Acetate&bull;EDTA) Agarose Gel Electrophoresis Buffer

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. The final solution should have a pH of ~8.5.

 *Unused or leftover acetic acid must be disposed of in a chemical waste bottle located in the fume hoods.* To avoid having leftover Acetic acid use a serological pipette to measure out the glacial acetic acid.

Working concentration is 1&times;, so measure 400 ml of 50&times; solution in graduated cylinder and then pour into 20 L carboy and fill to 20 L with ddH<sub>2</sub>0; if filling a 10 L carboy use 200 ml of stock.

---+++ 5&times; TBE Tris&bull;Borate&bull;EDTA Polyacrylamide Gel Electrophoresis

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 1&times; (for standard gels) or sometimes 0.5&times; (for PFGE). For 1&times;, measure 100ml of 5x solution into a 1L graduated cylinder and add ddH<sub>2</sub>O up to 500 ml to make it 1&times; or up to 1000 ml to make it 0.5&times;.

---+++ 6&times; Bromophenol Blue Agarose Gel Loading Buffer

| 24 ml | Glycerol |
| 0.015 g | Bromophenol Blue |
| 2.4 ml | 50&times; TAE loading buffer |
| to 40 ml | ddH<sub>2</sub>0 |

The final concentrations at 6&times; are 60% glycerol, 0.25% bromophenol blue, 3&times;TAE. At 1&times; are 10% glycerol, 0.0417% bromophenol blue, 0.5&times; TAE.

Note: Generally, your samples will be in PCR buffer that adds extra salt and buffering capacity. If you are adding pure DNA to this loading buffer, you may need to add PCR buffer to achieve consistent band mobilities.

<a id="ladder_working_solution"></a>
---+++ Agarose Gel DNA Ladder Working Solution

*To prepare the Invitrogen 1 Kb Plus ladder for loading on gels.* 

Create a 1:6 dilution of ladder combining it with Loading Buffer so that it reaches a 1&times; final concentration. 

| 166.6 ul | Ladder |
| 166.6 ul | 6&times; Loading Buffer (above) |
| 666.8 ul | Milipure H2O |

If you load 6 &micro;l of this Working Solution, it will contain 0.5 &micro;g of ladder and this image from the ThermoFisher website shows the number of ng of DNA in each band on the gel. You can compare the density of your bands to those of the ladded to estimate DNA concentrations.

<img src="https://www.thermofisher.com/TFS-Assets/BID/figures/10787018_10787026_Data.jpg-650.jpg" width="500">

---++ 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, SimonDAlton, JordanMonk, KateElston, LindseyWolf
Topic revision: r24 - 2023-09-20 - 16:31:05 - Main.IsaacGifford