Squish Buffer Recipe

10 mM Tris – pH 8.2

1 mM  EDTA

25 mM NaCl

1. Mix ingredients and autoclave or filter sterilize

TEG Buffer

25 mM Tris-HCl pH 8,
10 mM EDTA
50 mM Glucose, filter sterlized.

1. Combine Tris-HCl and EDTA solutions, autoclave.

2. Add filter-sterlized glucose.

TBE (Tris-Boric Acid-EDTA) – 20X stock solution

216 g Tris Base

18.6g EDTA

110g Boric Acid

1. Dissolve Tris in 700 ml of DI water, stir to mix.

2. After Tris is completely dissolved, add EDTA and mix. This may take a while.

3. After EDTA is completely dissolved, add Boric Acid and stir to mix.

4. Add DI water to 1000 ml. Filter using 1 L filter unit to remove particulates.

To make 0.5X TBE, dilute 25 ml of 20X stock in 1 L of DI water.

TE (10 mM Tris / 1mM EDTA)

0.6057 g Tris,

0.184 g EDTA

DI water

1. Add Tris to 100 ml of DI water and mix until dissolved in an autoclave-safe vessel

2. Add EDTA and mix to dissolve, adding additional DI water as necessary

3. Bring volume to 500 ml w/ DI water. Autoclave to sterilize

Low TE (10 mM Tris / 0.1mM EDTA)

0.6057 g Tris,

0.0184 g EDTA

500 ml DI water

1. Add Tris to 100 ml of DI water and mix until dissolved in an autoclave-safe vessel

2. Add EDTA and mix to dissolve, adding additional DI water as necessary.

3. Bring volume to 500 ml w/ DI water. Autoclave to sterilize

1% agarose, 500 ml

5 g Agarose

500 ml 0.5X TBE

1. Add agarose to TBE in a microwave safe container (ie, 1L bottle).

2. Microwave until TBE boils and agarose is completely dissolved, 5-7 minutes with lid partially off. Swirl to mix agarose at least once during microwaving, making sure to secure bottle cap prior to mixing.

3. Secure bottle cap, keep at 55-60° C until ready to cast gel.

LB Broth

10 g      Tryptone

10 g      NaCl

5 g        Yeast extract

1. Mix in 1 L autoclave bottle.

LB Agar

0.75 g  Agar

5 g       Tryptone

5 g       NaCl

2.5 g    Yeast extract

1. Mix in 1 L autoclave bottle.

2. Add DI water to 0.5 L and Autoclave. Cool to RT or 50° C if making plates for cloning. 

3. Add 10 ml of liquid LB agar to sterile Petri dish. Cool at store at 4° C.

X-Gal (50mg/ml)

200 mg  X-Gal (5-bromo-4-chloro-3indoyly-b-D-galactoside)

4 ml N,N,dimethylformamide

1. Mix X-Gal and N,N,dimethylformamide in 5 ml tube.

2. Cover tube with aluminum foil and store at -20° C.

IPTG (0.1 M)

0.9532 g IPTG

40 ml DI water

1. Add IPTG to DI water in a 50 ml tube. Filter sterilize.

Ampicillin (100 mg/ml)

50 mg ampicillin

50 ml DI water

1. Add ampicillin to DI water and filter sterilize.

2. Aliquot 1 ml in 1.5 ml tubes.

LB + amp + IPTG +Xgal plates (about 25 plates)

500 ml autoclaved LB agar

500 µl ampicillin (100 mg/ml)

50 µl X-gal (50 mg/ml) in N,N dimethylformamide

2.5 ml IPTG (0.1 M)

1. Melt LB Agar in microwave or use freshly autoclaved agar

2. Cool liquid agar to 50°C, preferably in a temperature controlled water bath.

3. Add ampicillin, X-gal, and IPTG, mix thoroughly.

4. Pour 10 ml into sterile Petri dishes. Cool plates, wrap in aluminum foil and store at 4° C.

Loding Dye (“OJ”)

0.25 g of Orange G

30 ml glycerol

70 ml DI water

1. Mix glycerol and water, autoclave.

2. Add Orange G, mix well. Store in 50 ml tubes at 4°C. Aliquot 1 ml in 1.5 ml tubes

dNTPs (10 mM)

37.5 µl dTTP, 100mM

37.5 µl dGTP, 100mM

37.5 µl dCTP, 100mM

1350 µl PCR-grade water

1. Mix all dNTP’s and DI water in a 2 ml tube.

2. Aliquot into 10 tubes of 150 µl ea.

SOC media

2 g          Tryptone

0.5 g       Yeast Extract

1 ml       1M NaCl

0.25 ml  1M KCl

1 ml       2M Mg2⁺ – filter sterlized

1 ml       2M Glucose – filter sterilized

1. Add Tryptone, yeast extract NaCl and KCl to 97 ml of DI water in an autoclave safe bottle. Autoclave.

2. Allow solution to cool to RT. Add 2M Mg2⁺ and 2M Glucose. Divide into 5 ml aliquots in sterile glassware.