Reagent and Buffer Recipes

General calculation resources

50x TAE

242 g Tris base
57.1 ml Glacial Acetic Acid
18.6 g EDTA
to 1 L ddH20

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).

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

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)

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

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

Makes ~4ml. Aliquot to 1.5 ml centrifuge tubes.

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

Dissolve 186.1 g NaEDTA 2 dH20 in 700 ml ddH20
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
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 –20C 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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Topic revision: r16 - 17 Jul 2017 - 14:49:39 - Main.SimonDAlton
 
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