Citric Acid – Na2HPO4 Buffer Preparation, pH 2.6–7.61

From Sigma Buffer Reference Center

Citric acid monohydrate, C6H8O7 • H2O, MW 210.14; 0.1 M contains 21.01 g/L.

Na2HPO4, MW 141.98; 0.2 M contains 28.40 g/L, or Na2HPO4 • 2H2O, MW 178.05; 0.2 M contains 35.61 g/L.

pH	x mL 0.1 M-citric acid	y mL 0.2-Na2HPO4
2.6	89.10	10.90
2.8	84.15	15.85
3.0	79.45	20.55
3.2	75.30	24.70
3.4	71.50	28.50
3.6	67.80	32.20
3.8	64.50	35.50
4.0	61.45	38.55
4.2	58.60	41.40
4.4	55.90	44.10
4.6	53.25	46.75
4.8	50.70	49.30
5.0	48.50	51.50
5.2	46.40	53.60
5.4	44.25	55.75
5.6	42.00	58.00
5.8	39.55	60.45
6.0	36.85	63.15
6.2	33.90	66.10
6.4	30.75	69.25
6.6	27.25	72.75
6.8	22.75	77.25
7.0	17.65	82.35
7.2	13.05	86.95
7.4	9.15	90.85
7.6	6.35	93.65

For 2021 QBoC

Need pH 3,4,5,6,7

Start with

• Citric acid (100 mM) (borrowed from P-Chem)
  • 2.65 g
  • 125 mL H2O
• Sodium phosphate dibasic (200 mM)
  • MW = 268.07
  • 200 mM = 8.042 g
  • 150 mL H2O

Then make

pH	x mL 0.1 M-citric acid	y mL 0.2-Na2HPO4
3.0	39.725	10.275
4.0	30.75	19.275
5.0	24.25	25.75
6.0	18.425	31.575
7.0	8.825	41.175

Test pH of 1:10, 1:2

• pH remains constant when diluted to 20% with LB.
• So make 50 mL of each diluted to 40% with LB. Students will dilute it 1:1 with LB
  • 20 mL buffer
  • 30 mL water
• Filter steriize, aliquot for students

Cnidocyte washing buffer

• 10 mM Tris
• 10 mM NaCl
• 10 mM EDTA

pH 7.6

To make	mL CWB
start with	mL water
mix	mL 1M Tris-base
and:	mL 5 M NaCl
and:	mL 0.5 M EDTA

Bring to pH 7.6, then final volume

Combine 400 ml of dH2O

0.6 g of Tris-HCl

0.29 g of NaCl

1.86 g of EDTA

Adjust the pH to 7.6 with 10 M NaOH.

Bring the volume to 500 ml with dH2O.

Store the buffer indefinitely at room temperature.

DNA Extraction Buffer for Gene & Genome

• 100 mM NaCl
• 50 mM Tris, pH8
• 25 mM EDTA
• 1% SDS

Make from stock solutions

To make	mL DNA Extraction Buffer
add:	mL 5M NaCl
add:	mL 1M Tris, pH 8
add:	mL EDTA 0.5M
add:	mL 20% SDS
add:	uL BMe AT THE LAST MINUTE!

Dilution buffer for NEB enzymes

Buffer composition:

Reagent	Final concentration
NaCl	300 mM
Tris-HCl	10 mM
DTT	1 mM
EDTA	0.1 mM
BSA	500 ug/mL
glycerol	50%
pH	7.4

To make 50 mL for use, make 25 mL of 2x, then mix 1:1 with glycerol

25 mL 2x Diluent B

Reagent	c1	c2	v1
NaCl	5M	0.3M	3 mL
Tris HCl	1M	0.01M	0.5 mL
DTT	1 M	1 mM	0.05 mL
EDTA	500 mM	0.1 mM	0.01 mL

plus BSA 25 mg

pH to 7.4, bring volume to 25, add glycerol to 50.

Filter sterilize

For quickie DNA extraction from Arabidopsis

• 200 mM Tris pH 7.5
• 250 mM NaCl
• 25 mM EDTA
• 0.5% SDS

To make	mL DNA Extraction Buffer
add:	mL 1M Tris, pH 7.5
add:	mL 5M NaCl
add:	mL EDTA 0.5M
add:	mL 20% SDS

Calcium-free HBS

Make from dry ingredients:

To make	mL Calcium-free HBS
start with	g MgCl2
add:	g KCl
add:	g NaCl
add:	g EGTA
add:	g HEPES

Compound	MW	mM	1000	500	350	250	100	mL
MgCl2(6H2O)	203.3	0.493	0.1	0.05	0.035	0.025	0.01	g
KCl	74.56	2.67	0.2	0.1	0.07	0.05	0.005	g
NaCl	58.43	137.9	8	4	2.8	2	0.8	g
EGTA	380.4	0.1	0.038	0.0192	0.0134	0.0095	0.00384	g
HEPES	238.21	10	2.383	1.1915	0.858	0.596	0.238	g

raise pH to 7.3 with NaOH

1X HBS (ca-) from stock solutions

CHECK THIS

Ingredient	stock M	mM	0.25	0.5	1	2	L
MgCl.6H2O	1	0.49	0.0001	0.0002	0.0005	0.0010	mL
KCl	1	2.67	0.0007	0.0013	0.0027	0.0053	mL
NaCl	5	137.90	0.0069	0.0138	0.0276	0.0552	mL
HEPES	1	10	0. 025	0. 050	0. 100	0. 200	mL

pH 7.3 with NaOH

filter sterilize

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Ingredient1	1X (mM)	10X (M)
CaCl2	0.9	0.009
MgCl2	0.493	0.00493
KCl	2.67	0.0267
NaCl	137.9	1.379
HEPES	10	0.1

Hank's Balanced Salt Solution

Gibco cat. #14025-092

stock bottle kept in tissue culture fridge

To make 1 Liter from dry ingredients

Components	MW	(mg/L)	mM
Calcium Chloride (CaCl2) (dihyd.)	147	185	1.26
Magnesium Chloride (MgCl2-6H2O)	203	100	0.493
Magnesium Sulfate (MgSO4-7H2O)	246	100	0.407
Potassium Chloride (KCl)	75	400	5.33
Potassium Phosphate monobasic (KH2PO4)	136	60	0.441
Sodium Bicarbonate (NaHCO3)	84	350	4.17
Sodium Chloride (NaCl)	58	8000	137.93
Sodium Phosphate dibasic (Na2HPO4-6H20)	268	90.6	0.338
D-Glucose (Dextrose)	180	1000	5.56

Filter sterilize

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To make from stock solutions:

Components	Ci (M)	cf (mM)	1000	500	250	mL
CaCl2	0.5	1.26	2.52	1.26	0.63	mL
MgCl2	1	0.493	0.493	0.247	0.108	mL
MgSO4	1	0.407	0.407	0.204	0.102	mL
KCl	1	5.33	5.33	2.67	0.131	mL
KH2PO4	1	0.441	0.441	0.22	0.11	mL
NaHCO3	0.5	4.17	8.34	4.17	0.209	mL
NaCl	5	137.93	27.59	13.79	6.9	mL
Na2HPO4	1	0.338	0.338	0.169	0.085	mL
Glucose	-	-	1	0.5	0.25	g

Filter sterilize

HEPES 1M

MW = 238.3

7.149 g / 30 mL

11.915 g / 50 mL

pH to 7.3 with NaOH pellets (~5 g/L)

Filter sterilize.

For fin clip, tail snip, or ear punch

• Obtain tissue 2 mm
  • tail snip
  • ear punch
  • fin clip
• Place tissue in 96 well plate
• Add 75 uL Alkaline Lysis Reagent for mouse, 50 uL for fish
• Heat to 95C 30 min (10 min to 1 hr)
• Cool to 4C
• Add 75 uL Neutralization Buffer for mouse, 50 uL for fish
• Use 3 uL (1-5) pre PCR reaction

Notes:

• DNA is suitable for PCR, not Southerns
• Heating longer than 30 min does not increase [DNA]
• pH of reagents does not need to be altered
• DNA yield is similar for tail snips and ear punches
• Too much tissue destroys PCR
• Store DNA at 4C or 20C

Make 1X PBS from 10X

100 mL 10X + 900 mL H2O

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10X PBS from Fisher

Fisher BP399-4 $51.07 as of 05/2020 ($34.45 January 2018!)

https://punchout.fishersci.com/shop/products/phosphate-buffered-saline-…

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10X PBS from stock solutions

from Sigma ready-made 1X:

M	ingredient
0.01	Na-K Phosphate
0.138	NaCl
0.0027	KCl

To make	L 10X PBS
add:	mL 5M NaCl
add:	mL 1M KCl
add:	mL 1M KH2PO4
add:	mL 1M Na2HPO4.7 H2O (MW = 268.07)

pH to 7.3 with NaOH

sterilize

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10X PBS from dry ingredients (Carrie's recipe)

To make	L 10X PBS
add:	g NaCl (MW = 58.44)
add:	g KCl (MW = 74.55)
add:	g KH2PO4 (MW = 136.09)
add:	g Na2HPO4.7 H2O

pH to 7.3 with NaOH

sterilize

0.1% Tween in PBS

PBS-Tw:	L
10x PBS:	mL
10% Tween 20 :	mL

PBS with:

• 0.1% Tween 20
• 0.2 g/L Na Azide (=0.02 %)

per Liter:

• 100 mL 10x PBS
• 10 mL 10% Tween 20
• 2 mL 10% Na azide

PBS-Tw-Az:	L
10x PBS:	mL
10% Tween 20 :	mL
10% Na azide :	mL

0.5% Triton X in PBS

To make	mL PBX
add:	10X PBS mL
and:	10% Triton-X mL

Plus water to final volume

PBX with 3% BSA for fish

To make	mL PBX/BSA
add:	g to PBX

0.5 M pH 6.7

(for transformation buffer for genetics)

MW (disodium salt) = 346.32

173.16 g/L

17.316 g/ 100 mL

Mix in ~80% of final volume

pH to 6.7 with 5M KOH

Filter sterilize

Aliquot

Freeze

1 M Potassium phosphate buffer

To make 100 mL of 1M potassium phosphate:

pH	mL 1M K2HPO4	mL 1M KH2PO4
5.8	8.5	91.5
6.0	13.2	86.8
6.2	19.2	80.8
6.4	27.8	72.2
6.6	38.1	61.9
6.8	49.7	50.3
7.0	61.5	38.5
7.2	71.7	28.3
7.4	80.2	19.8
7.6	86.6	13.4
7.8	90.8	9.2
8.0	94.0	6.0

from http://ivaan.com/protocols/151.html

To make approximately:	mL Potassium phosphate buffer
with:	pH
start with approximately:	mL 1M K2HPO4 (base)
and:	mL 1M KH2PO4 (acid)

Check the pH with the meter.

Add more K2 to raise the pH, or K1 to lower it.

Dilute as necessary to achieve the desired concentration.

To make 100 mL 1M sodium phosphate at a given pH:

pH	1 M Na2HPO4	1 M NaH2PO4
8.0	93.2 ml	6.8 ml
7.8	89.6 ml	10.4 ml
7.6	84.5 ml	15.5 ml
7.4	77.4 ml	22.6 ml
7.2	68.4 ml	31.6 ml
7.0	57.7 ml	42.3 ml
6.8	46.3 ml	53.7 ml
6.6	35.2 ml	64.8 ml
6.4	25.5 ml	74.5 ml
6.2	17.8 ml	82.2 ml
6.0	12.0 ml	88.0 ml
5.8	7.9 ml	92.1 ml

Dilute to the desired concentration.

From Sigma Buffer Reference Center

Sodium Acetate – Acetic Acid Buffer PREPARATION | pH 3.7–5.61 Sodium acetate trihydrate, CH3COONa • 3H2O MW 136.09; 0.2 M contains 27.22 g/L.

x mL 0.2 M-NaOAc and y mL 0.2 M-HOAc mixed

pH @ 18 °C	x mL 0.2 M-NaOAc	y mL 0.2 M-HOAc
3.7	10.0	90.0
3.8	12.0	88.0
4.0	18.0	82.0
4.2	26.5	73.5
4.4	37.0	63.0
4.6	49.0	51.0
4.8	59.0	41.0
5.0	70.0	30.0
5.2	79.0	21.0
5.4	86.0	14.0
5.6	91.0	9.0

(to neutralize acid wash during anti-5mC staining)

1L 1M

g	ingredient
61.83 g	Boric acid (H3BO3)
10 g	NaOH

water to 1L

100 mL 100mM

g	ingredient
0.6183 g	Boric acid (H3BO3)
0.1 g	NaOH

water to 100 mL

Tris 10 mM, EDTA 1 mM, pH 8

Make from stock solutions

To make	mL T10E1
add:	mL 1M Tris pH 8
add:	mL 0.5M EDTA

ingredient	cf		ci		25	50	mL
Tris pH 8	10	mM	1000	mM	0.25	0.5	mL
EDTA	1	mM	500	mM	0.05	0.1	mL

Tris 10 mM, EDTA 5 mM, pH 8

Make from stock solutions

To make	mL T10E5
add:	mL 1M Tris pH 8
add:	mL 0.5M EDTA

ingredient	cf		ci		25	50	mL
Tris pH 8	10	mM	1000	mM	0.25	0.5	mL
EDTA	5	mM	500	mM	0.25	0.5	mL

Tris-Acetate-EDTA buffer for agarose gel electrophoresis

BE Buffer 10x Stock Recipe:

• 108 g tris base.
• 55 g boric acid.
• 900 ml double-distilled H2O.
• 40 ml 0.5 M EDTA solution (pH 8.0)

To dilute Takara Ex-Taq

Store in freezer. Won’t freeze solid. Should keep forever.

• 50 μL Tween 20

• 50 μL Nonidet P-40 (anionic detergent = NP-40)

• 10 µL 1M DTT

• 1 mL KCl (1M)

• 200 μL Tris-Cl pH 8 (1M)

• 2 μL 0.5M EDTA pH 8

• 5 mL glycerol

• 4.2 mL ddH2O

To make	mL Taq dilution buffer
add:	mL Tris 1M pH 8
add:	mL KCl 1M
add:	mL EDTA 0.5M
add:	mL DTT 1M
add:	mL Tween 20
add	mL Nonidet P-40
add	mL glycerol

bring to final volume, filter sterilize in BSC, freeze