Common stock solutions

Protocols list 3/12/2015

10% Tween (500ml)

• Take 50ml of 100% Tween 20
• Add distilled water to bring final volume up to 500ml
• Filter sterilize

10% NP40 (500ml)

• Take 50ml of NP40
• Add distilled water to bring final volume up to 500ml
• Filter sterilize

1M Tris pH 7.5 or 8.0 (500ml)

5M =	1mol	0.5L	= 0.5mol
	1L
Tris	121.14g	0.5mol	= 60.57g
	1mol

• Dissolve 60.57g Tris Base in 150ml distilled water
• Use pH meter and titrate with 1M HCl until correct pH
• Add distilled water to bring final volume up to 500ml
• Send to be autoclaved

5M NaCl (500ml)

5M =	5mol	0.5L	= 2.5mol
	1L
NaCl	58.44g	2.5mol	= 146.1g
	1mol

• Dissolve 146.1g of NaCl in 150ml distilled water
• Add distilled water to bring final volume up to 500ml
• Send to be autoclaved

0.5M EDTA pH 8.0 (500ml)

0.5M =	0.5mol	0.5L	= 0.25mol
	1L
EDTA =	292.24g	0.25mol	= 73.06g
	1mol

• Dissolve 73.06g of EDTA in 150ml distilled water
• Use pH meter and titrate with 1M HCl or 10N NaOH until correct pH
• Add distilled water to bring final volume up to 500ml

Ampicillin Trihydrate Stock – 100mg/ml (50ml)

50 ml	100mg	1g	= 5g
	1ml	1000mg

• Dissolve 5g of Ampicillin Trihydrate in 25ml of distilled water
• Add 12 mL of 1M NaOH
• Add distilled water and bring final volume up to 50ml
• Filter sterilize
• Aliquot into 50, 1ml aliquots, store in -20 freezer in box

Ethidium Bromide 10mg/ml (10ml)

10 ml	10mg	1g	= 0.1g
	1ml	1000mg

• Dissolve 0.1g EtBr in 5ml of distilled water
• Add distilled water to bring volume up to 10ml
• Cover conical tube in foil

Proteinase K Storage Buffer

• Mix together 25ml of glycerol, 0.5ml Tris Solution pH 7.5, and 0.15mg CaCl2
• Add distilled water and bring final volume up to 50ml
• Filter sterilize

Proteinase K 10mg/ml (10ml)

10 ml	10mg	1g	= 100mg
	1ml	1000mg

• Dissolve 100mg of Proteinase K in 5ml of Proteinase K Storage Buffer
• Add Storage Buffer and bring final volume up to 10ml
• Aliquot into 10, 1ml aliquots, store in -20 freezer in box

Kanamycin Stock 25mg/ml (40ml)

40 ml	25mg	1g	= 1g
	1ml	1000mg

• Dissolve 1g of Kanamycin in 25ml of distilled water
• Add distilled water and bring final volume up to 40ml
• Filter sterilize
• Aliquot into 40, 1ml aliquots, store in -20 freezer in box

Chloramphenicol Stock 12.5mg/ml

40 ml	12.5mg	1g	= 0.5g
	1ml	1000mg

• Dissolve 0.5g of Chloramphenicol in 25ml of Ethanol
• Add Ethanol and bring final volume up to 40ml
• Filter sterilize
• Aliquot into 40, 1ml aliquots, store in -20 freezer in box

Potassium Acetate (KOAc) 500ml

3M =	3mol	0.5L	= 1.5mol
	1L
KOAc =	98.15g	1.5mol	= 147.225g
	1mol

• Dissolve 147.225g of KOAc into 200ml of distilled water
• Titrate with Acetic Acid to bring pH to 5.5
• Add distilled water and bring volume up to 500ml

2x HBS (500ml)

• Mix together 25ml of 50mM HEPES, 8g NaCl, and 0.14g of Na2HPO4•2H20 in 200ml of
  distilled water
• Use pH meter and titrate with 1M HCl or 10N NaOH until at pH 7.0
• Add distilled water and bring volume up to 500ml
• Aliquot into 50, 10ml aliquots and freeze at -20

2M CaCl2 (200ml)

2M =	2mol	0.2L	= 0.4mol
	1L
CaCl2•2H20 =	147g	0.4mol	= 58.80g
	1mol

• Dissolve 58.8g CaCl2•2H20 in 100ml of distilled water
• Add distilled water and bring volume up to 200ml
• Filter sterilize
• Aliquot into 20, 10ml aliquots and store in 4° fridge

10x TBST (1L)

• Dissolve 80.008g NaCl and 24.2g Tris Base in 600ml of distilled water
• Use pH meter and titrate with 1M HCl or 10N NaOH until at pH 7.6
• Add 100ml of 10% Tween
• Add distilled water and bring volume up to 1L

ACK Lysing Buffer (1L)

• Dissolve 8g of NH4Cl, 1g of KHCO3, and 0.04g of EDTA in 500ml of distilled water (use heat to
  help get EDTA into solution)
• Add distilled water and bring volume up to 1L
• Filter sterilize
• Store at 4°C

HBSS and 10mM EDTA

• Add 100ml 10x HBSS to 900ml distilled water and 2ml of 0.5M EDTA solution
• Filter sterilize
• Store at 4°C