Construction of BAC Libraries：SOLUTIONS FOR BAC LIBRARY CONSTRUCTION

SOLUTIONS FOR BAC LIBRARY CONSTRUCTION

Ingredient	Amount	Final Concentration
Trisma base	12.1 g	0.1 M
KCl	59.7 g	0.8 M
EDTA	37.2 g	0.1M
Spermidine-HCL	2.55 g	10 mM
Spermine-HCL	3.48 g	10 mM

Adjustment: pH to 9.4-9.5 with NaOH. Store at 4 °C.

1X Homogenization Buffer (HB): (1 liter)

Ingredient	Amount	Final Concentration
10X HB stock	100 ml	1X
Sucrose	171.15 g	0.5M

Store at 4 °C.
Adjustment: Before use add beta-mercaptoethanol to 0.15%.

20% Triton-X100 in 1 X HB: (100 ml)

Ingredient	Amount	Final Concentration
10X HB stock	10 ml	1X
Sucrose	17.12 g	0.5M
Triton X-100	20 ml	20%

Store at 4 °C.

Wash Buffer (1X Homogenization Buffer (HB) plus 0.5 % Triton X-100): (1 liter)

Ingredient	Amount	Final Concentration
10X HB stock	100 ml	1X
Sucrose	171.15 g	0.5M
Triton X-100 (20% stock in 1 X HB)	25 ml	0.5%

Store at 4 °C. Adjustment: Before use add beta-mercaptoethanol to 0.15%.

Na lauryl sarcosine (2%): (1 liter)

Ingredient	Amount	Final Concentration
Na lauryl sarcosine	20g	2%

Adjustment: Autoclave

Lysis Buffer: (1 liter)

Ingredient	Amount	Final Concentration
EDTA	500 ml of 1 M stock	0.5M (pH 9.0-9.3)
Na lauryl sarcosine	500 ml of 2% stock	1%<

Adjustment: Before use, add Proteinase K to 0.1 mg/ml

EDTA (0.5M, pH 8.0): (1 liter)

Ingredient	Amount	Final Concentration
EDTA	186.1 g	0.5 M
NaOH	20g

Adjustment: pH to 8.0 with NaOH or HCl. Autoclave.

EDTA (1.0 M, pH 9.0 to 9.3): (1 liter)

Ingredient	Amount	Final Concentratio
EDTA-di sodium salt	362.2 g	1.0 M
NaOH	40g

Adjustment: pH to 9.0 to 9.3 with NaOH. Autoclave.

Tris-HCl (pH 8.0): (1 liter)

Ingredient	Amount	Final Concentration
Trisma base	121.1 g	1M
HCl	42 ml
ddH20	800 ml

Adjustment: pH to 8.0 with HCl and adjust volume to 1 liter. Autoclave.

T10E1 (pH 8.0): (1 liter)

Ingredient	Amount	Final Concentration
Tris-HCl	10 ml of 1 M stock, pH 8.0	10 mM
EDTA	2 ml of 0.5 M stock, pH 8.0	1.0 mM

Adjustment: Autoclave

PMSF phenylmethyl sulfonyl fluoride

Caution: PMSF is a highly toxic acetylcholinesterase inhibitor. To avoid possible lab contamination, we purchase small 250 mg aliquots and then add isopropanol as above to the entire bottle to make a final concentration of 100 mM.

Ingredient	Amount	Final Concentration
PMSF	250 mg vial	100 mM
Isopropanol	14.35 ml

Store at 4 °C.

SOC Medium (for E. coli transformation): (1 liter)

Ingredient	Amount	Final Concentration
Bacto tryptone	20g	2%
Bacto yeast extract	5g	0.5%
NaCl	10 ml (1 M stock)	10 mM
KCl	2.5 ml (1 M stock)	2.5 mM
ddH2O	up to 980 ml	-

Adjustment: Adjust pH to 7.0, autoclave, and cool to room temperature and then add.



MgSO4/ MgCl2 Stocka	10 ml (2 M stock)	20 mM
Glucoseb	10 ml (2 M stock)	20 mM

a: Prepare and filter-sterilize a 2 M Mg stock ( 1 M MgCl2 6H2O 1 M MgSO4 - 7 H2O)
b: Prepare and filter-sterilize a 2 M Glucose stock.

LB Medium (E. coli ): (1 liter)

Ingredient	Amount	Final Concentration
Bacto tryptone	10g	1%
Bacto yeast extract	5g	0.5%
NaCl	10g	1%
Agar (for plates)	15g	1.5%

Adjustment: Adjust pH to 7.5. Autoclave.

Lysozyme solution: (100 ml)

Ingredient	Amount	Final Concentration
Glucose	0.9g	50 mM
EDTA (pH 8.0)	2 ml of 0.5 M stock	10 mM
Tris-HCl (pH 8.0)	5 ml of 1 M stock	25 mM
Lysozyme		5 mg/ml

Adjustment: Adjust volume to 100 ml with pure water and autoclave. Store at 4 °C. Add lysozyme just before use.

NaOH-SDS stock

Ingredient	Amount	Final Concentration
NaOH	1/2 volume of 0.4 N stock (8g/500 ml)	0.2 N
SDS	1/2 volume of 2% stock (10g/500ml)	1%

Adjustment: Mix NaOH and SDS stocks just prior to use.

Potassium acetate stock (pH 4.8-5.3): (100 ml)

Ingredient	Amount	Final Concentration
Potassium Acetate	60 ml of 5 M KOAc
Glacial Acetic Acid	28.5 ml
H2O	11.5 ml

20X SSC (pH 7.0): (1 liter)

Ingredient	Amount	Final Concentration
NaCl	175.3 g/l	3.0 M
Na Citrate	88.2 g/l	3.3 M

Adjustment: Dissolve in pure water and adjust pH to 7.0 with Citric acid.

50X TAE (Electrophoresis Buffer): (1 liter)

Ingredient	Amount	Final Concentration
Trisma Base	242 g
Glacial acetic acid	57.1 ml	2.5 M Tris-Acetate
EDTA	100 ml of 0.5 M EDTA (pH 8.0)	50 mM

Dilute 50 fold with pure autoclaved water.

5 X TBE (Electrophoresis buffer): (1 liter)

Ingredient	Amount	Final Concentration
Trisma Base	54 g	0.45 M
Boric Acid	27.5 g	0.45 M
EDTA	20 ml (0.5 M stock)	10 mM

The pH should be about 8.3 and should not require adjustment.

Mode of action: A bacteriostatic agent that interferes with bacterial protein synthesis by binding to the 50S subunit of ribosomes and preventing peptide bond formation.

Mechanism of resistance: The resistance gene specifies an acetylytransferase that acetylates and thereby activates the antibiotic.

Stock solution: 50 mg/ml in 100% ethanol. Store at -20 °C.

pBeloBAC11 plasmid preparation: 30 ug/ml

BAC transformation & minipreps: 12.5 ug/ml

Stock solution: 200 mg/ml in H2O. Filter sterilize and store at -20 °C.

Stock solution: 20 mg/ml in NN-dimethyl formamide. Store at -20 °C.

BAC transformation plates: 240 ml/plate (15 X 150 mm)

10X gel loading buffer: 500 ml

Ingredient	Amount	Final Concentration
Glycerol	350ml	35%
5X TBE	10ml	1X
0.5M EDTA (pH 8.0)	20ml	20mM
20% SDS	5ml	0.2%
10% bromophenol blue	30ml	0.6%
ddH2O	85ml	-