Difference: ProtocolsReagentRecipes (15 vs. 16)

Revision 162017-07-17 - SimonDAlton

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META TOPICPARENT name="ProtocolList"

Reagent and Buffer Recipes

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General calculation resources

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50x TAE

242 g Tris base
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18.6 g EDTA
to 1 L ddH20
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Prepare by filling bottle with 900 ml of ddH20 and adding the above chemicals. Adjust volume to 1 L with ddH20.
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Prepare by filling bottle with 700 ml of ddH20 and adding the above chemicals. Adjust volume to 1 L with ddH20.
  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 ddH20; 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).
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5x TBE Tris•Boric Acid•EDTA

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5x TBE Tris•Boric Acid•EDTA

 
54 g Tris base
27.5g Boric Acid
20 mL 0.5 M EDTA (pH 8.0)
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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
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Cool the solution, and neutralize by adding 120 μl of 1 M Tris (pH 8.0)
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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

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  1. Dissolve 1 g of desired NTP in 10 ml ddH2O:
    • ATP - Adenosine 5′-triphosphate disodium salt (MW 551.14)
    • GTP - Guanosine 5′-triphosphate sodium salt hydrate (MW 523.18)
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  1. pH to 8.0 with 1 M NaOH. It takes approximately 1.0–1.5 ml.
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  1. pH to 8.0 with 1 M NaOH. It takes approximately 1.0–1.5 ml.
 
  1. Add ddH2O to final volume of:
    • GTP - 18.14 ml
    • GTP - 19.11 ml
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  1. Store at –20°C in 1.0–1.5 ml aliquots.
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  1. Store at –20°C in 1.0–1.5 ml aliquots.
 
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HEPES•NaOH, 1M, pH 7.0

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HEPES•NaOH, 1M, pH 7.0

 pH buffer with less temperature dependence than Tris.

To make 100 ml:

 
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