Standard reagent recipes

DNA gel loading buffer

Composition of DNA gel loading buffer (from Maniatis) is:
15% Ficoll
0.25% Bromophenol Blue
0.25% Xylene cyanol FF
Notes: Apparently BBP is worth omitting completely and 0.25% is an
overestimate of the amount of XC needed. Some recipes use 10mM Tris
pH7.5, I omit this. I suggest 0.125% Xylene cyanol FF and 0.05%
bromophenol blue.
note
Cyanol FF runs at 4kb,
bromphenol blue runs at 300bp,
Orange G runs at 50bp

Shrimp alkaline phosphatase

37C for 10 minutes and then 65C for 15 minutes

IPTG

make up to 0.5M in water - this is a 1000x stock for protein expression

Chloramphenicol

make up 1000x in ethanol to stock solution of 10mg/ml
Working strength is 10micrograms/ml
store at -20C

Ampicillin

make up in water to stock solution of 100mg/ml and sterile filter
this is 1000x - use at 100micrograms/ml
store at -20C

Kanamycin

Make up in water to stock solution fo 25mg/ml and sterile filter
this is 500x use at 50micrograms/ml
store at -20C

Tetracycline

Make up in 50%v/v ethanol and water mixture to 12.5mg/ml. Sterile filter
and store in the dark at -20C
this is 1000x use at 12.5micrograms/ml

nb for pCDM7 vectors use 25micrograms/ml ampicillin and 7.5micrograms/ml
tetracycline

Expression cell antibiotics

Note for Rosetta Gami and other BL21 expression cells
use antibiotics at following concentrations
kanamycin 15 micrograms/ml
chloramphenicol 34 micrograms/ml
tetracycline 12.5 micrograms/ml
rifampicin 200 micrograms/ml

Competent cells reagents

TFB1: mix for 500mls
30mM KOAc Sigma P5708 1.47g
50mM MnCl2 4.95g
100mM KCl 3.72g
10mM CaCl2 0.55g
15% v/v Glycerol 75mls
make up to 500mls and sterile filter, store at 4C

TFB2: mix for 500mls - note only worth making 100 or 250mls
10mM Na-MOPS pH7.0 - 5mls of 1M NaMOPS pH7.0
75mM CaCl2 4.16g
10mM KCl 0.37g
15% v/v glycerol 75mls
make up to 500mls and sterile filter, store at 4C

note protocol is 500ml cells to od 0.6 spin in 100mls cold TFB1 then 20ml
cold TFB2 then to -80C

Ethidium bromide

stock solution is 10mg/ml
use at 500microg/ml ie 50microlitres per litre of TBE or TAE

TBE

2.5 litres of 10x TBE
270g Tris base
127.5g Boric acid
18.6g of EDTA
water to 2.5litres
Autoclave

TAE

1litre of 50xTAE
242 g Tris base
57.1mls acetic acid
1/10 mole of EDTA ie 37.2g of Na2EDTA 2H2O

TBE

Final concentration of tween 20 is 0.5%

TYP

For 1 litre
16 g Bacto Tryptone
16g Yeast Extract
5g NaCl
2.5g K2HPO4

LB

1 litre
10g Bacto Tryptone
5 g bacto yeast extract
10g NaCl

For plates add 15g of bacto agar to the litre of LB medium before
autoclaving. Remember to let it cool to about 50C before adding
antibiotics. Flame with a Bunsen burner to remove bubbles.

Tissue Culture

FCS - heat inactivate by heating at 56øC for 30 mins. Freeze at -20C in
25ml aliquots.
Glutamine freeze aliquots of 5mls of 200mM glutamine
Penicillin and streptomycin - freeze aliquots of 2.5mls of ,000 U/ml
penicillin and 10mg/ml streptomycin mix

R10 or D10 mix for 500mls
add 50mls of FCS to give 10% FCS
add 5mls of 200mM Glutamine to give 2mM Glutamine
- add 2.5mls of 10,000 U/ml penicillin and 10mg/ml streptomycin mix to
give 50U/ml penicillin and 50U/ml streptomycin


Note Trypan blue stains dead cells 

Freezing mix

Freeze in 90%FCS and 10%DMSO
Make 20% DMSO, 80%FCS and resuspend cells in half vol of 100%FCS then add
20%DMSO, 80%FCS

DEPC water

Add 5 ml DEPC to 5 liters distilled water. Stir overnight.
Autoclave for 30 minutes.

Thrombin

make thrombin stock up as 1000 cleavage unit per ml in PBS
use 20microliters per ml of final reaction volume

Protein loading buffer 5x 

50mls 1M Tris pH 6.8
30g SDS
1g bromophenol blue
100mls glycerol
make up to 250mls with water
for reducing buffer add 100microlitres of beta2me to 400microlitres of
this

Protein loading buffer 5x 

75g Tris base
470g Glycine
25g SDS
make up to 2.5litres with water

Ammonium persulphate (APS)

make a 10% solution in water
usually just make 0.1g upto 1ml
or 0.2g upto 2mls with water

Protein gels 

15% gel mix
46mls water
150mls 1.0M Tris pH 8.8
2mls 20% SDS
For 1 gel use 4.9ml of this plus
5mls of 30% acrylamide (protogel National Diagnostics)
0.1mls of 10% APS (ammonium persulphate, sigma)
4microlitres of TEMED (Sigma)

12% gel mix
84mls water
150mls 1.5M Tris pH 8.8
2mls 20% SDS
For 1 gel use 5.9ml of this plus
4mls of 30% acrylamide (protogel National Diagnostics)
0.1mls of 10% APS (ammonium persulphate, sigma)
4microlitres of TEMED (Sigma)

Stacking gel mix
171.25mls water
31.5mls 1.0M Tris pH 6.8
1.25mls 20% SDS
For 1 gel use 2.04ml of this plus
415 microlitres of 30% acrylamide (protogel National Diagnostics)
25 microlitres of 10% APS (ammonium persulphate, sigma)
2.5microlitres of TEMED (Sigma)

Coomassie stain 

1.25g Coomassie brilliant blue
250ml methanol
50ml acetic acid
make up to 550ml with water and filter
- detection limit is around 0.1-0.5 micrograms

Destain for Coomassie stain 

1 litre
100mls acetic acid
300mls methanol
make up to 1 litres with water

Slow Coomassie stain 

Coomassie stain slow, but needing minimal destaining
50% methanol
0.05% v/v Coomassie brillian tblute R-250 (Bio Rad or Pierce)
10% acetic acid
40% water
dissolve Coomassie blue in methanol first then add acetic acid and water.
filtere if necessary.

NP40 lysis buffer 

1% NP40 in PBS with azide and filtered)
note IGEPAL is equivalent to Nonidet NP40
With protease inhibitors (leupeptin, pepstatin, PMSF, EDTA, +/- aprotinin)

RIPA buffer 

50 mM Tris-Cl, pH 8.0
150 mM NaCl
0.1% SDS
0.5% Sodium deoxycholate
1% NP40 (IGEPAL is NP40)
Protease inhibitors
Note – Variants include using Tris pH 7.4, adding 1mM EDTA, or using 1% Triton X100 instead of 1% NP40

Aggressive lysis buffer 

0.15 M NaCl
5 mM EDTA, pH 8
1% Triton X100
10 mM Tris-Cl, pH 7.4
Just before using add: 1:1000 5 M DTT
1:1000 100 mM PMSF in isopropanol
1:1000 5 M e-aminocaproic acid

Western Blotting Transfer buffer 10X 

30.3 g Trizma base (= 0.25 M)
144 g Glycine (= 1.92 M)
make up to 1L with water
pH should be 8.3; do not adjust

To make 1 L of 1x Blotting buffer:
200 ml Methanol
100 ml 10x Blotting buffer
700 ml water

TBS (1X)

20mM Tris pH 7.4,
500mM NaCl
- note can make a 10X stock

TBS-Tween (TBST) 

TBS-Tween (TBST)
20 mM Tris-HCl, pH 7.4
500mM NaCl
0.05% Tween 20
note Tween 20 is difficult to pipette, so take the appropriate volume from a 10% working
stock solution

Tween 20 

Make a 10% stock for easy pipetting

Blocking solution - Western blots 

3% nonfat dried milk (Marvel) or BSA in TBST (see recipe above)
For 100mls use 3g Milk or BSA in 100mls TBST

PBSA 

PBSA
PBS with 0.03% azide
add 3mls of 10% azide to each litre

Western blot stripping solution

100 mM 2-mercaptoethanol
2% SDS
62.5 mM Tris-Cl pH 6.7
- Put the blot in stripping buffer, incubate 50 degrees for 30 min. in a
sealed plastic container in the shaking water bath. (this can be repeated
if necessary. Rinse with TBST. Can be stored at 4 degrees.Reblock with
TBSTM as in step 2 of Western probing method (above) before reprobing
with antibody.

10X DNA loading buffer

For 100mls
20%w/v Ficoll 400 20g
0.1M NaEDTA pH 8.0 20mls of 0.5M
1% w/v SDS 1g or 10mls of 10%
0.25% w/v bromphenol blue 0.25g

Oligonucleotides 

When an oligo is delivered, write the number of the oligo on its lid
and put it in the oligo box in the -20 freezer.
Our standard stock solution of oligos is 100pmol/microliters
If oligos are supplied dry, look for the number of nmoles supplied
(usually about 50nmoles).
Add 10x this volume of milliQ autoclaved water to the tube.
ie if there are 49.2nmoles, add 492 microlitres.

For PCRs, make a small aliquot of a working solution of 10pmol/microliter
by a further 10x dilution of the stock.
eg. Take 2microliters of the stock and add 18microliters of water to make
the total volume up to 20 microliters.
If you want to add 10pmol to your PCR you just add 1 microlter of
this working solution.
If you want to add 20pmol to your PCR you just add 2 microlters of
this working solution.

PCR

dNTPs use 5ul of 10mM dNTPs in 50ul reaction
note this is 10mM total or 2.5mM of each NTP
use 20pmols of each oligo

Xgal and IPTG

use at 2%
- make Xgal up in DMF

Triton Wash 

To make 1litre
0.5% Triton X100 5mls of Triton X100
50mM Tris pH8 50mls of 1M
100mM NaCl 25mls of 4M
0.1% Azide 10mls of 10%
make up to 1000mls with water

Resuspension buffer 

To make 1litre
50mM Tris pH8 50mls of 1M
100mM NaCl 25mls of 4M
make up to 1000mls with water

MES  

(morpolino ethanesulfonic acid)
Titrate 1M solution to appropriate pH - usually 6.5 with NaOH
Store in 2.5ml aliquots at -20C

Sodium acetate

Make stock of 0.2M acetic acid (11.55mls in 1000mls)
Make 0.2M sodium acetate solution
Mix 41mls of 0.2M acetic acid with 9mls of 0.2M sodium acetate
- the result should be 0.2M sodium acetate pH4.0, but always check with a pH meter. 

DTT 

Make up to 1M in 10mM Sodium acetate pH 5.2 and freeze at -20C

Protease inhibitors

PMSF - serine protease inhibitor with a half-life in water of 15-60 minutes
make up to 0.1M stock in isopropanol and store at -20C
- this is a 1000x stock so use at 0.1mM PMSF

Pepstatin - acid protease inhibitor
make up a 1mg/ml stock solution in methanol
Store at -20C in aliquots
This is a 1000x stock use at 1microgram/ml

Leupeptin - serine and cysteine protease inhibitor
make up a 1mg/ml stock solution in PBS
Store at -20C in aliquots
This is a 1000x stock use at 1microgram/ml

Aprotinin - serine protease inhibitor
make up at 1mg/ml stock solution in PBS
Store at -20C in aliquots
This is a 1000x stock use at 1microgram/ml

Propidium iodide  Protein

(Sigma P4170 100mg )
make up to 50micrograms per ml in H20 or PBS and use at
concentration of about 10microg/ml
peak excitation is at 536nm, peak emission at 620nm, both are broad
stain for about 30mins to an hour at 10micrograms/ml

Diptheria toxin  

(Calbiochem 322326) 1mg $135 or sigma ? D?
make 1000x of 1mg/ml in DMEM store aliquots at -20C

Hygromycin  

(Sigma H777-2 ?650 1)g
get powder make to 60mg/ml in DMEM 1.25ml
aliquots at -20C final conce for Phoenix onx-e cells 375ug/ml

Puromycin  

Sigma powder 1000x is 1mg/ml in PBS

Polybrene  

(Hexadimethrine bromide) Stock solution is 8mg/ml (0.1g in
12.5mls). Make this up in RPMI 1640 and filter then aliquot and freeze at -20C.
This is 1000x so working solution is 8micrograms/ml

Geneticin  

G418 Gibco/BRL #11811-098 25g 50mg/ml in 0.1M Hepes pH 7.5

mouse IL3 

mouse interleukin 3 - 100,000x dilute in RPMI to 1000x 500ul aliquots

Antibodies and Fluorescent reagents

Generally, secondary antibody used at 25micrograms/ml and use
20microlitres for staining
Jackson anti-Fc is 1.4mg/ml when made up in 0.5mls
- use at 50microg/ml and use 20microl of this

Streptavidin PE
- use 20microL of 50microg/ml

Extravidin PE
- multimerisation
use 0.312mg of extravidin PE per mg of 48kDa protein
use proportionately less for smaller proteins to preserve molar
ratios
Extavidin PE is supplied at 0.2mg/ml

AKTA tubing

0.5mm internal diameter tubing vol is 0.2microliters/mm
0.75mm internal diameter tubing (green) vol is 0.44microliters/mm
1.0mm internal diameter tubing (brown) vol is 0.79microliters/mm

Ultrafiltration membranes

Amicon/Millipore
wash in 0.1M sodium hydroxide for 30 minutes then rinse extensively in
distilled water.

Surface plasmon resonance reagents

Sigma streptavidin (S-4762) 1mg, made up to 0.5mg/ml in 10mM sodium acetate pH4.0 and aliquoted (100 microliters)
Ethanolamine 1M ethanolamine HCl adjusted to pH 8.5 with NaOH

HBS-EP 10mM HEPES pH7.4, 150mM NaCl, 3mM EDTA, 0.005% (v/v) surfactant P20 or Tween 20
To make 1 litre of 10x HBS-EP
   100mls of 1M HEPES pH 7.4
   375mls of 4M NaCl
   60mls of 0.5M EDTA
   0.5mls (= 500ul) of surfactant P20 or Tween 20

Amine coupling kit
NHS and EDC - to both of these add 10mls filtered de-ionised water, replace cap, agitate until solids are dissolved then put 100ul aliquots in Eppendorfs 
label Eppendorf tubes containing EDC with E and put in a plastic clip-lock bag. Store at -20C 
label Eppendorf tubes containing NHS with N and put in a plastic clip-lock bag. Store at -20C 
Store Ethanolamine at 4C

 

General stock solutions 

10% azide
4M NaCL
4M MgCl2
10M Urea