Dr. Zamir K. Punja Laboratory

Lab binder Media and Buffer Anabel Crouse (Sep. 2010)

1) Liquid Media

Potato dextrose broth

12.0 g potato dextrose broth
500 ml dH₂O

Add potato dextrose broth and water to a 1000 ml flask

Mix

Cap with cotton batting (wrapped in cheesecloth) and foil

Label with autoclave tape and autoclave

2) Solid Media

Potato dextrose agar

19.5 g PDA
500 ml dH₂O

Add PDA and water to a 1000 ml flask

Mix

Cap with cotton batting (wrapped in cheesecloth) and foil

Label with autoclave tape and autoclave.

N.B. – ANY required antibiotic should be added to media after autoclaving.

V8 agar

141.5 ml V8 juice (shake well before pouring)
358.5 ml dH₂O
10.0 g Bacto-agar

Add V8, water and agar to 1500 ml flask

Mix

Cap with cotton batting (wrapped in cheesecloth) and foil

Label with autoclave tape and autoclave

2) Solid Media con’t

Modified VDYA – BCNB (per L)

150. 0 mL carrot juice
1 g CaCO₃
2 g glucose
2 g yeast extract
1.0 g oxgall
15.0 g Bacto-agar

Add carrot juice, CaCO₃, glucose, yeast extract, oxgall and stir bar to 2 L beaker.

Add enough dH₂O to bring volume to 1 L

Adjust to pH 5.2

Add Bacto-agar and above liquid to a 2 L flask

Cap with cotton batting (wrapped in cheesecloth) and foil

Label with autoclave tape and autoclave

Once autoclaved add the following:

0.5 g PCNB (in 2.0 mL chloroform)
30 mg neptatin (added as a powder)
100 mg streptomycin sulfate
2 mg chlortetracycline – use 0.2 ml of 10 mg/ml stock
2 mg Botran – use 2 ml of 1 mg/ml stock
20 mg Ridomil – use 2 ml of 10 mg/ml stock

Medium 523 (per L)

10.0 g sucrose
8.0 g Casein hydrolysate
4.0 g yeast extract
2.4 g K₂HPO₄ 3H₂O
0.3 g MgSO₄ 7H₂O
15.0 g Bacto-agar

Add sucrose, Casein hydolysate, yeast extract, K₂HPO₄ 3H₂O and MgSO₄ 7H₂O to a 1 L beaker

Add enough dH₂O to bring volume to 1 L

Adjust to pH 7.0

2) Solid Media con’t

TB- CEN media (per L)

150 ml carrot juice
1.0 g CaCO₃
15.0 g Bacto-agar

Add carrot juice and CaCO₃ to a 1 L beaker

Add enough dH₂O to bring volume to 1 L

Add Bacto-agar and above liquid to a 2 L flask

Cap with cotton batting (wrapped in cheesecloth) and foil

Label with autoclave tape and autoclave

Once autoclaved add the following:

0.5 g PCNB (dissolved in 2.0 ml chloroform)
8.5 mg mystatin – use 2 ml solution
500.0 mg streptomycin sulfate
30.0 mg chlortetracycline – use 3.0 ml
1.0 mg Botran – use 1 ml of 1 mg/ml stock
10.0 Ridomil – use 1 ml of 10 mg/ml stock

Modified Lochwood

150.0 ml carrot juice
1.0 g CaCO₃
2.0 g yeast extract
1.0 g oxgall
15.0 g Bacto-agar

Add carrot juice, CaCO₃, yeast extract and oxgall to a 1 L beaker

Add enough dH₂O to bring volume to 1 L

Adjust to pH 5.2

Add Bacto-agar and above liquid to a 2 L flask

Cap with cotton batting (wrapped in cheesecloth) and foil

Label with autoclave tape and autoclave

Once autoclaved add the following:

1.0 g PCNB (dissolved in 4 ml chloroform)
50.0 mg neptatin, 10 mL solution
250.0 mg chloramphenicol
60.0 mg penicillin

MS Media (per L)

30.0 g sucrose
0.18 g MgSO₄
1.7 g K₂HPO₄
1.9 g KNO₃
1.65 g NH₄NO₃
0.33 g CaCl₂

MS Vitamins

0.1 g myoinositol
1.0 ml thiamine solution (0.8 mg/ml)

MS Micronutrients

10.0 ml solution A (10X)
1.0 ml solution B (500X)

Solution A (10X)

0.0062 g/L H₃BO₃
0.0223 g/L MnSO₄ H₂O
0.0086 g/L ZnSO₄ H₂O
0.0278 g/L FeSO₄ 7H₂O
0.0373 g/L Na₂EDTA

Solution B (500X)

0.025 mg/l NaMoO₄ 2H₂O
0.025 mg/l CuSO₄ 5H₂O
0.025 mg/l CoCl₂ 6 H₂O
0.83 mg/l KI

Procedure to prepare 1 L MS Media:

Add the preweighed dry ingredients into a 100 ml beaker

Note: do not cross contaminate chemicals; thoroughly clean spatula

on a fresh tissue; discard excess chemical, do not put back in

jar

2) Add micronutrient and vitamin solutions

3) Add dH₂O until volume is just less than 1000 ml

4) Add magnetic stir bar to beaker and place on electric stirrer

* While chemicals are dissolving standardize the pH meter

5) Adjust to pH 5.8 (use small amounts of 1 N KOH)

6) Pour solution into 1000 ml graduated cylinder and enough dH₂O to

bring volume to 1000 ml

7) Pour 500 ml of MS solution into two 1000 ml flasks

8) Add 1% agar per flask (5 g per 500 ml MS solution)

9) Cap with cotton batting (wrapped in cheesecloth) and foil

10) Label with autoclave tape and autoclave

11) Cool until the media feels very warm. In the laminar flow hood aseptically add 100 mg/l ampicillin powder to each flask. Mix

N.B. – regular Petri plates hold 25 ml media and deep Petri plates

hold 40 ml media

TBM – 2RB A

Add 200 g cut up carrots to 350 ml dH₂O and blend in Waring Blender on high for 1-2 minutes

2) Filter carrot mush through cheesecloth. Squeeze pulp dry. Add a

small amount of pulp to the juice.

3) In a separate beaker add 20 g of lab grade agar (NOT Bacto-agar)

and 2 g yeast extract (cell culture type) to 500 ml dH₂O

4) Adjust pH of each solution to pH 5.0 using 1 N H₂SO₄

5) Pour carrot juice into a 2000 ml Erlynmeyer flask, cap with cotton

batting (wrapped in cheesecloth) and foil.

6) Label with autoclave tape and autoclave

7) Pour agar solution into a 1000 ml flask, cap with cotton batting

(wrapped in cheesecloth) and foil.

8) Label with autoclave tape and autoclave

Once the carrot juice has been autoclaved & cooled add the following:

1.0 g PCNB (dissolved in 2-5 ml chloroform)
1.0 g oxgall
0.06 g Nystatin (dissolved in 5-10 ml methanol)
0.06 g penicillin
0.25 g chloramphenicol

1.5% water agar

For 500 ml:

7.5 g agar
add enough dH₂O to bring volume to 500 ml
autoclave

Bavendamm’s medium (for Tyrosinase detection)

For 1000 ml:

Solution A

15 g Difco malt extract
20 g Difco Bacto-agar
add enough dH₂O to bring volume to 900 ml
autoclave

Solution B

5 g tannic acid/gallic acid in 100 ml. Adjust to pH 4.5 with NaOH and

autoclave.

Mix the two autoclaved solutions (A and B) and store in the dark.

Ginseng and Carrot Suspension Media (per 1.5 L)

1) Fill beaker ¾ full with dH₂O.

2) Add the following nutrients:

macronutients 75 ml of 20X (or 150 ml of 10X)
micronutrients 1.5 ml
calcium 75 ml of 20X
Fe-EDTA 7.5 ml
MS vitamins 1.5 ml

3) Add sugars and stir with magnetic stir bar until clear

Myo-inositol 0.15 g
Sucrose 45 g

4) Pour solution into a graduated cylinder and add enough dH₂O to bring

volume to 1.5 L

5) Divide solution: pour 0.5 L into a 500 ml Erlenmeyer flask to make carrot

Dissolve:

18.62 mg/100 ml

22.1 mg/ 100 ml

MS ½ D

6) Add plant growth regulators to carrot MS ½ D solution:

NAA 2.5 mM 2.5 ml/l (3.75 ml/1.5 L)
2,4-D 2.3 mM 2.3 ml/l (3.45 ml/1.5 L)

7) Standardize pH meter with pH 4 buffer

8) Adjust the pH of both solutions to pH 5.8

9) Double foil the caps & stick on autoclave tape

10) Autoclave on liquid 20 cycle

* solid 8 g/L agar

TBE (Tris/Borate/EDTA) is a buffer solution containing a mixture of Tris base, boric acid and EDTA.

In molecular biology, TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solution are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. EDTA is a chelator of divalent cations, particularly magnesium. As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of EDTA is to protect the nucleic acids against enzymatic degradation. But since magnesium is also a co-factor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases, its concentration in TBE or TAE buffers is generally kept low ( ~ 1mM)

1 L of 5X stock solution

54 g of Tris base (CAS # 37186)
27.5 g of boric acid (CAS # 11280)
20 mL of 0.5 M EDTA (CAS # 60004) (ph 8.0)

TBE should be diluted to 0.5 X prior to use in electrophoresis.