Difference: ProtocolsReagentRecipes (20 vs. 21)

Revision 212023-08-30 - JeffreyBarrick

 
META TOPICPARENT name="ProtocolList"

Reagent and Buffer Recipes

General calculation resources

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TrisAcetateEDTA"> 50x TAE Tris•Acetate•EDTA

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Gel Electrophoresis

 
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TrisAcetateEDTA Agarose G"> 50× TAE Tris•Acetate•EDTA Agarose Gel Electrophoresis Buffer

 Used as a buffer for 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 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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>		Prepare by filling bottle with 700 ml of ddH20 and adding the above chemicals. Adjust volume to 1 L with ddH20. The final solution should have a pH of ~8.5.
 
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<		 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.
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>		 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.
 
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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.
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>		Working concentration is 1×, so measure 400 ml of 50× solution in graduated cylinder and then pour into 20 L carboy and fill to 20 L with ddH20; if filling a 10 L carboy use 200 ml of stock.
 
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TrisBorateEDTA"> 5x TBE Tris•Borate•EDTA

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TrisBorateEDTA Polyacrylam"> 5× TBE Tris•Borate•EDTA Polyacrylamide Gel Electrophoresis

  Used as a buffer for Polyacrylamide Gel Electrophoresis.

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 (for standard gels) or sometimes 0.5x (for PFGE). For 1x, measure 100ml of 5x solution into a 1L graduated cylinder and add ddH2O up to 500 ml to make it 1x or up to 1000 ml to make it 0.5x.
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>		Working concentration is 1× (for standard gels) or sometimes 0.5× (for PFGE). For 1×, measure 100ml of 5x solution into a 1L graduated cylinder and add ddH2O up to 500 ml to make it 1× or up to 1000 ml to make it 0.5×.
 
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20x SB Agarose Gel Buffer (Sodium Borate)

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20× SB Agarose Gel Buffer (Sodium Borate)

  An alternative buffer for Agarose Gel Electrophoresis.

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.

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6x Bromophenol Blue Gel Loading Buffer

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>		Note: This is not commonly used in our lab!
 
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30 ml Glycerol
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6× Bromophenol Blue Agarose Gel Loading Buffer

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0.01 g Bromophenol Blue
to 50 ml ddH20
 
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DNA Ladder with Loading Dye

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24 ml Glycerol
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0.015 g Bromophenol Blue
2.4 ml 50× TAE loading buffer
to 40 ml ddH20
 
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>		The final concentrations at 6× are 60% glycerol, 0.25% bromophenol blue, 3×TAE. At 1× are 10% glycerol, 0.0417% bromophenol blue, 0.5× 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.

Agarose Gel 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 ~4 ml. Aliquot to 1.5 ml centrifuge tubes.
  To prepare the Invitrogen 1kb+ ladder: Create a 1:6 dilution of ladder in water, accounting for the addition of your dye of choice (see example below)

166.6 ul Ladder
166.6 ul 6x Loading Buffer (above)
666.8 ul Milipure H2O

Stock solutions

Tris-HCl, 1 M

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 (pH 8.0)

Dissolve 186.1 g Na•EDTA•2H20 (EDTA disodium salt dihydrate) in 700 ml ddH20
Adjust pH to 8.0 with 10 M NaOH ( It should take ~ 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
*Warning: Add NaOH pellets slowly! Dissolving is very, very exothermic.

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
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
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 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

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
  4. 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 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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