Cook Molecular Lab

Chemical Solutions


ACK Lysis Buffer (Groves)

Recipe:
4.01 g NH4Ac
0.5 g KHCO3
0.01 g EDTA
Bring volume to 500 ml with sddH2O.

Storage: 4°C

Disposal: Sink



1.5% Agarose Gel

Warning: Contains ethidium bromide, a powerful mutagen.

Recipe:
1.5 g Agarose
100 ml 1X TBE

1. Mix in 200 ml Erlenmeyer flask.  Swirl gently.
2. Loosely cover the flask top with a Kim wipe plug.  Capping or plugging the top may cause the contents of the flask to explode during heating.
3. Heat to boiling in microwave (approximately 2 minutes).
4. Wear cloth gloves and swirl the flask gently.
5. Continue until the agarose is well dissolved.
6. Add 10 ml 10 mg/ml EtBr.

Storage: Room temperature

Disposal: Ethidium Bromide Waste container in lab



5% Chelex

Recipe:
5 g Chelex Resin
In 100 ml volumetric flask, bring volume to 100 ml with sddH2O.
Store in 100 ml GIBCO bottle with stir bar.

Storage: 4°C

Disposal: Sink



10X dNTP

Recipe:
10 µl dATP (100 mM)
10 µl dCTP (100 mM)
10 µl dGTP (100 mM)
10 µl dTTP (100 mM)
60 µl sddH2O

Storage: -20°C.  Thaw on ice just prior to use.

Disposal: Sink



Ethidium Bromide (EtBr)

Warning:  Ethidium bromide is a powerful mutagen.

Recipe: Final concentration of 10 mg/ml in sddH2O.

Storage: 4°C

Disposal: Ethidium Bromide Waste Container (located in fume hood)

Decontamination of Ethidium Bromide Solutions

1. Add 1 g of powdered activated charcoal for each 1 L of solution.

2. Store the solution for 1 hour at room temperature, shaking it intermittently.

3. Filter the solution through a Whatman No. 1 filter, and discard the filtrate (and filter) as EtBr solid waste.

4. Contact the Physical Plant for waste disposal.  Fill out the appropriate forms.



Field Buffer

Recipe:
38 g Mannitol
24 g Sucrose
50 ml 1M Tris-HCl, pH 7.5
20 ml 0.5M EDTA.
Bring volume to 1 L with sddH2O.

Storage: 4°C

Disposal: Sink



Homogenization Buffer

Recipe:
85.58 g Sucrose
5 ml 1 M Tris, pH 8.0
1 ml 0.5 M EDTA, pH 8.0
In 500 ml volumetric flask, bring volume to 500 ml with sddH2O.

Storage: 4°C

Disposal: Sink



Loading Dye for Agarose Gels

Recipe:
0.025 g Bromophenol Blue
4 g Sucrose
Bring volume to 10 ml with sddH2O.

Storage: 4°C

Disposal: Sink



Phenol

Warning:   Phenol is an extremely caustic chemical which will cause severe burns.  Exposure may be through rapid absorption of tissue or through inhalation.  All work with phenol must be conducted under the fume hood.  Protective clothing will be a lab coat, apron, gloves, and goggles. In the event of an exposure, rinse the affected area immediately with running water for at least 10 minutes.

Recipe:
Only redistilled and buffered phenol are used for nucleic acid extractions since the oxidation products of phenol may damage the DNA.
1. Remove distilled phenol from the freezer and allow to warm to room temperature.
2. Melt crystals in 65°C waterbath.  Do not place the hot bottle on a cool counter, as it may result in the bottle fracturing.
3. Add 0.1g of 8-hydroxyquinoline to a 250 ml dark bottle with stir bar.
 The 8-hydroxyquinoline acts as an antioxidant and will turn the phenol yellow.
4. Avoid any splashing and gently pour in 100 ml of melted phenol.
5. Add 100 ml of 1M Tris-HCl, pH 8.  Stir gently for 5 minutes.
6. Allow phases to separate for 5 minutes.
7. Gently decant the aqueous (top) phase into a waste container.
8. Repeat steps 5 to 7.
9. Add 100 ml of 0.1M Tris-HCl, pH 8.  Repeat two more equilibration runs with 0.1M Tris-HCl until the pH of the aqueous phase is 8.
10. Dispose after 2 months.  Before each use, ensure that the pH>7.  If pH<7, repeat steps 5 to 9.

Storage: 4°C

Disposal: Phenol Waste Container (located in fume hood).



Phosphate Buffered Saline, PBS (Groves)

Recipe:
1.08 g Na2HPO4, 7H2O
0.53 g Na2HPO4
0.53 g NaCl
0.1 g KH2PO4
0.1 g KCl
Bring volume to 500 ml with sddH2O.

Storage: 4°C

Disposal: Sink



Primer (10 mM)

Recipe: Depends on the stock concentration for specific primer.  Refer to previous calculations of other primers

Storage: -20°C

Disposal: Sink



Primer (2.5 mM) - for cycle sequencing

dilute the 10 mM primer by 1/4

Recipe:
100 ml of 10 mM primer
300 ml of SDD water

Storage:  -20°C

Disposal: Sink



Primer (1:100 dilution, or 0.1 mM)

Recipe:
2 ml of 10 mM primer
198 ml of sddH2O

Storage: -20°C

Disposal: Sink



Proteinase K

Recipe: 10 mg/ml in sddH2O

Storage: -20°C



RNase A

Recipe:
10 mg/ml in sddH2O.  Heat to 100°C for 15 minutes and allow to cool slowly to room temperature.  Aliquot in 1 ml units.

Storage: 4°C



10X Taq PCR Buffer (Kocher)

Recipe:
670 µl 1M Tris, pH 8.0
67 µl 1M MgCl2
10 µl 10% Tween 20
253 µl sddH2O

Storage: -20°C

Disposal: Sink



20X TAE, pH 7.4

Warning:  Acetic acid is a corrosive chemical.

Recipe:
54.4 g Sodium acetate trihydrate
96.9 g Tris
7.44 g EDTA
40 ml Acetic Acid, Glacial
Bring volume to 1L with ddH2O.

Storage: Room temperature

Disposal: Sink



10X TBE, pH 8.3

Recipe:
108 g Tris
55 g Boric Acid
9.3 g EDTA
Bring volume to 1L with ddH2O.

Storage: Room temperature

Disposal: When a white precipitate is visible in the bottom of the bottle.  Sink.



Tissue Lysis Buffer (MVZ)

Warning: Contains ß-mercaptoethanol

Recipe:
5 ml 1M Tris-HCl
10 ml 0.5M EDTA
10 ml 10% SDS
2 ml 5M NaCl
1 ml ß-mercaptoethanol
Bring volume to 100 ml with sddH2O.

Storage: 4 °C

Disposal: Biohazard Waste Container (located in fume hood).



TE (Tris-EDTA)

Recipe:
1 ML 1M Tris, pH 8
0.2 ml 0.5M EDTA, pH 8
Bring volume to 100 ml with sddH2O.

Storage: 4°C

Disposal: Sink



T(1/10)E

Recipe:
100 µl 1M Tris, pH 8
2 µl 0.5M EDTA, pH 8
9898 µl sddH2O.
Final volume will be 10 ml.

Storage: 4°C

Disposal: Sink



10% SDS

Recipe: 10 g SDS
In 100 ml volumetric flask, bring volume to 100 ml with sddH2O.

Storage: 4°C

Disposal: Sink



STE

Recipe:
5 ml 5M NaCl
5 ml 1M Tris, pH 8.0
1 ml 0.5 M EDTA, pH 8.0
In 500 ml volumetric flask, bring volume to 500 ml with sddH2O.

Storage: 4°C

Disposal: Sink