Buffers

Buffers kdorfman Tue, 06/16/2009 - 17:51

Here are some commonly made buffers for biochemistry and molecular biology

HBSS

Sigma Buffer Reference Center

Citric acid buffer

Citric acid buffer kdorfman Tue, 11/16/2021 - 20:03

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

DEB

DEB kdorfman Mon, 12/19/2011 - 17:40

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!

Diluent B

Diluent B kdorfman Mon, 09/26/2022 - 20:21

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

Edwards Extraction Buffer

Edwards Extraction Buffer kdorfman Tue, 03/22/2022 - 13:20

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

HBS (Ca-)

HBS (Ca-) kdorfman Tue, 12/27/2011 - 17:53

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


HBS/Ca/Mg

HBS/Ca/Mg kdorfman Tue, 12/13/2011 - 20:32
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

  1. Concentrations from Lab 9, Bioimaging 2008, Dave Gross ↩︎

10X HBS (dry)

10X HBS (dry) kdorfman Fri, 08/09/2013 - 18:55

10X HBS/Ca/Mg from dry ingredients

To make mL 10X HBS/Ca/Mg
add: g CaCl2.2H20 (MW = 147.02)
add: g MgCl.6H2O (MW = 203.3)
add: g KCl (MW = 74.56)
add: g NaCl (MW = 58.43)
add: g HEPES (MW = 238.31)

pH 7.3 with NaOH

Filter sterilize

10X HBS (stocks)

10X HBS (stocks) kdorfman Fri, 08/09/2013 - 18:56

10 X HBS from stock solutions

To make mL 10X HBS/Ca/Mg
add: mL 1M CaCl2.
add: mL 1M MgCl
add: mL 1M KCl
add: mL 5M NaCl
add: mL 1M HEPES

pH 7.3 with NaOH

Filter sterilize

1X HBS (stocks)

1X HBS (stocks) kdorfman Fri, 08/09/2013 - 20:22
To make mL 10X HBS/Ca/Mg
add: mL 1M CaCl2.
add: mL 1M MgCl
add: mL 1M KCl
add: mL 5M NaCl
add: mL 1M HEPES

pH 7.3 with NaOH

Filter sterilize

HBSS

HBSS kdorfman Fri, 04/03/2020 - 14:57

Hank's Balanced Salt Solution

Gibco cat. #14025-092

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



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

HEPES kdorfman Tue, 12/20/2011 - 14:16

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.

HotSHOT genomic DNA prep

HotSHOT genomic DNA prep kdorfman Tue, 06/21/2022 - 15:00

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

Alkaline Lysis Reagent

Alkaline Lysis Reagent kdorfman Tue, 06/21/2022 - 15:07

Alkaline Lysis Reagent for HotSHOT DNA prep

Recipe from Mike Charles 10/15/03; mikchar@umich.edu; retrieved from Craig Albertson's lab

NOTE: Re calculate for 1 M NaOH - 10 M will damage your plastic tube!!

Reagent stock conc final conc amt
NaOH 10 N 25 mM 125 uL
EDTA 0.5 M 0.2 mM 20 uL
dI water to final volume 50 mL

Calculator

To make mL Alkaline Lysis Reagent
add: uL 10M NaOH
add: uL 0.5M EDTA
plus to final volume: mL H2O

pH =~12

Neutralization Buffer

Neutralization Buffer kdorfman Tue, 06/21/2022 - 15:09

Neutralization Buffer for HotSHOT DNA prep

Dilute stock Tris-HCl to 40 mM

pH =~5

PBS

PBS kdorfman Tue, 06/16/2009 - 17:52

Make 1X PBS from 10X

100 mL 10X + 900 mL H2O


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-…


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


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

from stock solutions

from stock solutions kdorfman Wed, 11/13/2013 - 18:13

10X PBS from stock solutions

from Sigma ready-made 1X:

M (1X) M (10X) ingredient
0.01 0.10 Na-K Phosphate
0.138 1.38 NaCl
0.0027 0.027 KCl
calculated from Carrie's dry ingredients recipe:
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


OR

To match Sigma recipe:

Make 1M Na2HPO4 (base); 1 M KH2PO4 (acid)

Mix to pH 7.3

(should be ~38.25 mL Na2HPO4, 11.5 mL KH2PO4) from the Sigma Buffer Reference Center

pH mL Na Phos dibasic mL Na Phos monobasic
7.2 36.0 14.0
7.4 40.5 9.5

Then

To make L 10X PBS
add: mL 5M NaCl
add: mL 1M KCl
add: mL 1M K-Na Phosphate

PBX (for fish)

PBX (for fish) kdorfman Mon, 03/23/2020 - 13:57

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/BSA

PBX/BSA kdorfman Tue, 11/10/2020 - 18:44

PBX with 3% BSA for fish

To make mL PBX/BSA
add: g to PBX

PIPES

PIPES kdorfman Thu, 01/19/2012 - 16:43

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

Potassium phosphate

Potassium phosphate kdorfman Sat, 01/14/2012 - 04:58

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.

Sodium Phosphate

Sodium Phosphate kdorfman Mon, 12/12/2011 - 19:20

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.

Sodium acetate buffer

Sodium acetate buffer kdorfman Tue, 11/16/2021 - 19:53

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

Sodium borate buffer

Sodium borate buffer kdorfman Fri, 11/05/2021 - 14:10

(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

T10E1

T10E1 kdorfman Mon, 12/19/2011 - 17:54

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

T10E5

T10E5 kdorfman Mon, 12/19/2011 - 18:25

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

TAE

TAE kdorfman Tue, 06/16/2009 - 17:52

Tris-Acetate-EDTA buffer for agarose gel electrophoresis

1X TAE

1X TAE kdorfman Tue, 06/16/2009 - 19:02
To make L 1X TAE
add: mL 50X TAE

plus water to final volume

50X TAE stock

50X TAE stock kdorfman Tue, 06/16/2009 - 19:01

Buy from Fisher TAE 50x, pH 8.3, 4L, part #BP13324

from Maniotis

To make mL 50X TAE
add: g Tris base
add: mL glacial acetic acid
add: mL 0.5M EDTA
OR add: g EDTA

pH to 8 with acetic acid or NaOH

should this be 8.4?

Ingredient cf (mM) 1000 750 600 500 mL
Tris 2000 242 181.5 145.2 121 g
Acetic acid 1000 57.1 42.9 34.3 28.6 mL
EDTA 0.5 M 50 100 75 60 50 mL
OR
EDTA 50 18.62 13.965 11.172 9.31 g

pH to 8 with acetic acid or NaOH

Maniotis says to autoclave, but the salt concentration is so high that nothing will grow in it if you don't.

Taq dilution buffer

Taq dilution buffer kdorfman Fri, 05/31/2013 - 17:21

To dilute Takara Ex-Taq

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

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