Difference: ProtocolsReagentRecipes (15 vs. 16)

Revision 162017-07-17 - SimonDAlton

 
META TOPICPARENT name="ProtocolList"

Reagent and Buffer Recipes

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General calculation resources

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General calculation resources

 
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50x TAE

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50x TAE

 
242 g Tris base
57.1 ml Glacial Acetic Acid
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.
 
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<		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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>		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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TrisBoric AcidEDTA"> 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 ddH20
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<		Working concentration is 1x, so measure 100ml of 5x solution in a 1L graduated cylinder and add ddH2O up to 500 ml to make it 1x. Note: Used as a buffer for Polyacrylamide Gel Electrophoresis.
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>		Working concentration is 1x, so measure 100ml of 5x solution in a 1L graduated cylinder and add ddH2O up to 500 ml to make it 1x. Note: Used as a buffer for Polyacrylamide Gel Electrophoresis.
 

20x SB Agarose Gel Buffer (Sodium Borate)

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38.2 g Borax
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38.2 g Borax
 
10 g Boric Acid
to 1 L ddH20

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 ddH20 to make it 1x.

6x Bromophenol Blue Gel Loading Buffer

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30 ml Glycerol
0.01 g Bromophenol Blue
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30 ml Glycerol
0.01 g Bromophenol Blue
 
to 50 ml ddH20

DNA Ladder with Loading Dye

375 ul Loading Buffer (above)
250 ul Ladder
325 ul 10x PCR Buffer
3 ml ddH20
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<		Makes ~4ml. Aliquot to 1.5 ml centrifuge tubes.
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>		Makes ~4ml. Aliquot to 1.5 ml centrifuge tubes.
 

Stock solutions

Tris-HCl, 1 M

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121 g Tris base in 800 ml ddH20
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121 g Tris base in 800 ml ddH20
 
Adjust to pH 8.0 with HCl
Mix and add ddH20 to 1 L

EDTA, 0.5 M

Dissolve 186.1 g NaEDTA 2 dH20 in 700 ml ddH20
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Adjust pH to 8.0 with 10 M NaOH (~ 50ml)
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Adjust pH to 8.0 with 10 M NaOH (~ 50ml)
 
Mix and add ddH20 to 1 L

NaOH, 10 M

Dissolve 400g NaOH in 450 ml ddH20
Mix and add ddH20 to 1 L

Potassium acetate, 5 M

29.5 ml glacial acetic acid
KOH pellets to pH 4.8 (several)
ddH20 to 100 ml
Store at room temperature

NaCl, 1M

Dissolve 58.4 g of NaCl in 800 ml ddH20
Mix and add ddH20 to 1 L

CaCl2, 1 M

Dissolve 110.9 g of CaCl2 in 800 ml ddH20
Mix and add ddH20 to 1 L
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Aliquot into 2 500 ml bottles and autoclave
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Aliquot into 2 500 ml bottles and autoclave
 

MgSO4, 1 M

Dissolve 120.3 g of MgSO4 in 800 ml ddH20
Mix and add ddH20 to 1 L
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Aliquot into 2 500 ml bottles and autoclave
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Aliquot into 2 500 ml bottles and autoclave
 
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RNase A, 5 mg/ml

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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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<		For in vitro transcription or deoxyribozyme reactions:
  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)
  2. pH to 8.0 with 1 M NaOH. It takes approximately 1.0–1.5 ml.
  3. Add ddH2O to final volume of:
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>		 For in vitro transcription or deoxyribozyme reactions:
  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)
  2. pH to 8.0 with 1 M NaOH. It takes approximately 1.0–1.5 ml.
  3. 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

pH buffer with less temperature dependence than Tris.
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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 ddH2O.
  2. pH to 7.0 with 6 M NaOH. It takes approximately 2.0 ml.
  3. Add ddH2O to final volume of 100 ml.
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