Tris-HEPES-SDS Running Buffer (10X)
Recipe for 10X Tris-HEPES-SDS Buffer:
Tris Base 121 g
HEPES 238 g
SDS 10 g
Add ultrapure water to 1,000 ml
1X Tris-HEPES-SDS Running Buffer consists of:
•100 mM Tris
•100 mM HEPES
•3 mM SDS
1X Tris-HEPES-SDS Running Buffer is used to run Precise Protein Gels
(Pierce), and compatible with Tris-Glycine SDS sample loading buffer.
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10 X Phosphate Buffered Saline (PBS) Recipe
Dissolve the following in 800ml distilled H2O.
80g of NaCl
2.0g of KCl
26.8g of Na2HPO4•7H2O
2.4g of KH2PO4
Adjust pH to 7.4.
Adjust volume to 1L with additional distilled H2O.
Sterilize by autoclaving.
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50X TAE Recipe
1)Add the following to 900ml distilled H2O
242g Tris base
57.1ml Glacial Acetic Acid
18.6 g EDTA
2)Adjust volume to 1L with additional distilled H2O
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Carbenicillin-10ml(1,000x Stock)
100 mg/ml carbenicillin in water
Reagents needed:
1 g carbenicillin disodium salt
10 ml ddH2O
Directions:
1) Dissolve 1 g of carbenicillin into 10 ml of ddH2O.
2) Filter through a 0.22 µm filter to sterilize.
3) Aliquot and store at -20°C.
4) Use at 1:1000 dilution in LB or LB-Agar.
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Gels for Western Blots
|
Ingredient
|
Stacking Gel
|
|
|
Resolving
Gel |
|
|
|
|
|
Acrylamide% |
|
|
|
|
|
6% |
8% |
10% |
12% |
|
40 % Acrylamide/bisacrylamide
|
0.625 ml |
1.5 ml |
2.0 |
2.5 |
3.0 |
|
Tris Buffer (pH 6.8
or 8.8) |
0.625 ml (6.8) |
2.5 ml (8.8) |
2.5 |
2.5 |
2.5 |
|
dH2O |
3.65 ml
|
5.8 ml |
5.3 |
4.8 |
4.3 |
|
10% SDS
|
50 ul
|
100 ul |
100 |
100 |
100 |
|
u10% Ammonium
Persulfate (APS) |
50 ul
|
100 ul
|
100 |
100 |
100 |
|
TEMED
|
5 ul |
8 ul
|
8 |
8 |
8 |
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Preparing Gels/Electrophoresis
To make gel, mix 2% (3g) agarose with 150 ml TAE buffer and heat in microwave until clear.
Add 2 ul of red gel dye, insert combs, and pour into mold. Solidifies in ~1 hour.
Remove gel mold and place into chamber with negative pole at top (adjacent to wells).
Cover gel with TAE buffer.
Make template indicating what sample each well contains.
Add ~ 3 ul drop xylazine cyanol gel dye to side of all PCR tubes.
Mix gel dye into sample, and add entire sample (20ul) to each well, Run on Volts Constant (105-120 V) for ~ 1 hour.
Run from negative to positive pole.
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Tissue Prep for PCR
Collect tissue in 1.5 ml eppendorf tube and freeze until ready.
Boil beaker of water in microwave and then put on hot plate (#4-5) and maintain water at a low boil.
Add 50 ul NaOH solution (25 mM NaOH + 0.2 mM EDTA) to each sample.
Push tissue down into solution with pipet tip, place in water bath racks, and boil for 15-30 min (watch that lids don’t pop off tubes – if they do remove water from tube and add new 50 ul of solution).
Neutralize by adding 50 ul of 40 mM Tris-Cl (0.628 g Tris-Cl/100 ml water), pH 5, and vortex.
Can freeze samples at this stage if you like.
PCR Mix Ingredients
Per reaction:
1) nuclease-free water 14.2 ul
2) Buffer 1.6 ul
3) Taq DNA polymerase 0.2 ul
4) dNTPs 0.1 ul
5) Primer 0.2 ul
6) DNA template 1.0 ul
Mix enough of 1 through 5 for all your reactions (add enough for 2 more tubes ) in 1.5 ml Eppendorf tube.
Label enough tubes for all reactions and add 16.3 ul of this master mix to each PCR tube.
Add 1 ul of DNA template to each PCR tube (add into master mix or into drop on side of tube).
Place caps on PCR tubes and Invert tubes twice to mix.
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