Keeney lab home

Solutions and Buffers

Ammonium Persulfate, 10%

mix in 15 ml polypropylene conical tube:

1 g Ammonium persulfate
10 ml water

Store at 4C. Make up fresh each month.

Buffer B/EDTA, 10x

for 500 ml:
25 ml 3 M KCl
25 ml 2M Hepes/KOH
50 ml 0.5 M EDTA

to 500 ml with water, autoclave

Buffer B/EDTA, 1x

15 mM KCl
10 mM Hepes/KOH, pH 7.8
5 mM EDTA

carbenicillin

50 mg/ml (1,000X) [50 mg/ml final]
1 g in 20 ml. Filter sterilize. Aliquot 500 ml/eppendorf

Coomaissie Blue destain
(for protein gels)
45% methanol; 10% acetic acid

for 1L:
450 ml methanol
100 ml acetic acid
add water to 1 L

Coomaissie Blue Stain
(for protein gels)
0.1 % Coomaisse;45% methanol; 10% acetic acid

for 200 ml: (make up in a glass stoppered bottle)
0.2 g Coomaissie Blue R
90 ml methanol
20 ml acetic acid

can be reused-pour back into bottle

Denhardt's, 100x

1.6 g Ficoll
1.6 g polyvinylprop
1.6 g BSA (bovine serum albumin)
water to 80 ml, filter, store at -20C

DNA markers
-100 bp ladder
-1 kb ladder

concentration is on printed on tube
dilute to 0.1 ug/ul with 1/20th volume loading dye
(example: a tube contains 200 ul at 0.5 ug/ul. Add 750 ul water and 50 ul loading dye for a total volume of 1 ml)
aliquot 50 ul per microfuge tube, label, and put in box in -20C freezer

DTT, 1M
(dithiothreitol)

don't weigh out-add water to bottle to make 1 M-6.5 ml/g
200 ml aliquots into eppendorfs, store at -20C

EDTA, 0.5M
pH 8.3

93.1 g EDTA (disodium salt, dihydrate) FW=372.24
into 450 ml water
pH with 10 M NaOH-will take ~30 ml
EDTA will not dissolve completely until pH is 8.3
bring up to 500 ml, autoclave

ethidium bromide

5 mg/ml (10,000X)
mutagen-wear gloves

Hepes/KOH, 2M
pH 7.8

238.3 gm/500 ml
pH with 10 M KOH, autolave

IPTG
(200 mg/ml)

for 1 ml:
0.2 g IPTG (isopropylthio-b-D-galactoside)

weigh out IPTG (TOXIC! wear gloves, don't breath dust!) on analytical balance in B311into an eppendorf or a polypropylene 15 ml conical. Dissolve in 800 ml water, then adjust the volume to 1 ml final. Filter sterilize with a syringe. Aliquot 100 ml/eppendorf, store at -20C in box labeled x-gal, IPTG

KCl, 3M

111.84g/500 ml
autoclave

KOH, 10M

56.1 g/100 ml water
caustic-wear glove
store in plastic bottle, not glass

LiAc, 1M

51 g LiAc
add water to 500 ml
aliquot 100 ml/bottle and autoclave

loading dye, 10x

for 20 ml:
8 gm sucrose (40% final)
10 ml 0.5 M EDTA (250 mM final)
add water to 20 ml, dissolve sucrose completely
0.15 gm bromphenol blue (1.5% final)
1 ml aliquots into eppendorfs-store at -20C

lysozyme (for STET prep)

10 mg/ml in STET buffer
weigh out 100 mg lysozyme into a 15 ml conical
Add STET buffer to the 10 ml mark, mix by inverting until dissolved
Aliquot 200 ul/microfuge tube, put in container on door of -20C freezer

MOPS buffer, 10x

for 500 ml:
23.1 g MOPS
3.4 g Sodium Acetate
0.93 g EDTA (disodium salt)
dissolve in ~450 ml water
adjust to pH 7.0 with 10M NaOH, bring up to 500 ml total

PEG 3350, 44%

PEG 3350 (Sigma P-3640) 110 g
add water to 250 ml
filter sterilize, aliquot 50 ml/bottle into sterile bottles

PMSF, 100 mM
(phenylmethylsulfonylfluoride)

0.17g/10 ml isopropyl alcohol
make up in polypropylene Falcon tube, store at -20C

Sequencing
bottom solution

WEAR GLOVES!
75 ml 40% acrylamide (19:1)
125 ml 10x TTE
230 g UREA
50 g sucrose
25 mg bromphenol blue
Add water to 500 ml
Filter sterilize, store at 4C

Sequencing
top solution

WEAR GLOVES!
75 ml 40% acrylamide (19:1)
25 ml 10x TTE
230 g UREA
Add water to 500 ml, store at 4C

SDS, 10%

10 g in 100 ml water
do not autoclave
detergent-do not breath dust when weighing out

Southern Denaturation Buffer

8 g NaOH
35 g NaCl
add water to 1L total volume

Southern Hybridization solution
6x SSC
5x Denhardt's
10 mM EDTA
0.5% SDS

for 250 ml:
75 ml 20X SSC
12.5 ml 100X denhardt's
5 ml 0.5 M EDTA
1.25 g SDS
water to 250 ml, aliquot, store at -20C

Southern Neutralization Buffer

for 1 L:
87.75 g NaCl
60.5 g Tris base
add water to ~900 ml
pH to 7.4 with HCl, top off to 1 L

SSC, 20X

for 1 L:
175.5 g NaCl
88 g Na citrate
add water to 1L total volume, pH to 7.5 (takes very little!)

STET buffer

recipe is at the bottom of the "STET plasmid prep" protocol in the protocols section

stop buffer (sequencing)

9.5 ml deionized formamide
400 ul 0.5 M EDTA
50 ul of 10% bromphenol blue/10% xylene cylanol (0.5 % final)

sucrose, 20%

100 g sucrose
50 ml 10X buffer B/EDTA
add water to 500 ml total volume, filter sterilize

sucrose, 30%

150 g sucrose
50 ml 10X buffer B/EDTA
add water to 500 ml total volume, filter sterilize

sucrose, 70%

350 g sucrose
50 ml 10X buffer B/EDTA
add water to 500 ml total volume, filter sterilize

TA, 10X

(tris-acetate buffer)

for 1 L:
60.55 g Tris Base
11.25 ml acetic acid
use pH tape to confirm is 8.0-8.5

TE, 100x

for 500 ml:
60.6 g TRIS base
37.2 g disodiumEDTA
add water to ~400 ml
pH with HCl to 7.6 (will take ~25 ml)adjust to 500 ml and check pH
aliquot 100 ml/bottle, autoclave

Tris/HCl, 2M

for 500 ml:
121.1 gm TRIS base
400 ml water
pH with HCl, then top off to 500

TTE, 10x

for 5L:
537.5 g Tris base
177.5 g taurine
9.25 g EDTA

X-gal
(20 mg/ml)

for 1 ml:
20 mg x-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside)
1 ml N,N-dimethylformamide (DMF)
weigh out x-gal (wear gloves-irritant) on analytical balance in B311into an eppendorf or a polypropylene 15 ml conical. DMF is on shelf below the hood. Dissolve thoroughly. Aliquot 200 ml /tube and store in box labeled x-gal, IPTG in -20C

Keeney Lab Home