Ampicillin Stock 100g/L

3g ampicillin sodium salt (Fisher BP1760-25)

Add sterile water to raise volume to 30 ml. Filter sterilize through 0.22um syringe filter. Then aliquot into 1 ml and store at –20C.

3M Ammonium Acetate, pH 5.2, 100ml

24.6g ammonium acetate anhydrous (FW 82.03). Dissolve in about 60 ml 3M acetic acid. Stir. Warm. Stir.

When cooled to room temperature, adjust pH to 5.2.

Autoclave

For 100ml 24.6g

200ml 49.2

300ml 73.8

Bradford Assay

1. Make up protein standard dilutions as follows:

A) 2 ul of BSA stock + 98 ul H20

B) 4 ul of BSA stock + 96 ul H20

C) 6 ul of BSA stock + 94 ul H20

D) 8 ul of BSA stock + 90 ul H20

BSA stock is 2.0 mg/ml H20

2. Make up protein sample dilutions as follows:

For crude lysate and diluted supernantant:

A) 2 ul protein + 98 ul H20

B) 5 ul protein + 95 ul H20

C) 10 ul protein + 90 ul H20

For protein fractions and pools

B) Same as above

D) 15 ul protein + 85 ul H20

3. Add 0.9 ml of Bradford Reagent to each eppie tube, votex each tube for a few seconds.

4. Take 2 readings of each tube at 595 nm

5. Then do the calculations.

Bradford Assay Calculation Sheet

1. Protein Standard

Tube mg. Protein OD at 595 nm

A 0.004

B 0.008

C 0.012

D 0.016

E 0.020

2. Plot the standard curve ,OD at 595 nm vs mg of protein standard. Attach graph to back of prep record

3. Next do calculations that determine the protein concentration of your samples:

Sample A: OD at 595 nm = X

Match ‘X’ up the correct mg of protein that was found from the standardization curve. Divide this amount by the amount of protein which was assayed.

Buffer SP-A (HIV-1 NC Prep) (RD)

Stock Add

50 mM Tris-HCL pH 8.0 FW 121.1 6.06 g

10% glycerol Liq. 100 ml

0.1 M NaCl FW 58.44 5.8 g

0.1 mM ZnCl₂ 0.2 M 0.5 ml

10 mM BME 14.4 M 0.694 ml

Buffer SB

Stock Add for 1 L

20mM NaPO₄ pH 6.1 1 M 20 ml

1 M NaCl FW 58.44 58.44 g

10 mM BME 14.4 M 0.714 ml

Blue/ Green Dye

80% Formamide 8 ml

Xylene Cyanol FF 10mg

Bromophenol Blue 10mg

10 mM EDTA (stock 0.5 M

pH 8.0) 200 ul

fill with water to: 10 ml

Camera DS34

Coomassie Stain: Light box computer room 125 22

Et. Br. Gels: Light box in dark room 4.5 max 1 sec

Chloramphenicol 34g/L

Fisher Stock store at room temperature

EtOH solution store at –20C

1.02 g chloramp.

To 30 ml with EtOH

1 M Calcium Chloride

100 ml

CaCl₂ Anhydrous FW 110.99 11.10 g

CaCl₂·2H2O FW 147.02 14.70 g

No need to sterilize. Divide into 10 ml aliquots. Store in –20 C

Coomassie Blue R-250 Staining solution

(From Bio-Rad for Tris-Glycine gels)

500 ml

40 % MeOH 200 ml

10 % Acetic Acid 50 ml

Coomassie Blue R-250 0.5 g

Fill to 500ml with water.

Competent Cells (protocol for one strain)

Day one:

Prepare 100 ml LB with agar for 2 plates.
Autoclave:

100 ml LB

Small beaker for TSS solution

(extra flasks if preparing more than one strain

Small centrifuge tubes

Pour 2 plates LB agar with NO antibiotic (except for strains containg pLys, both S and E, plasmid-then add 34 g/L chloramphenicoll)
Remove stock cells from –70 C freezer and place on ice to thaw
Streak two plates, label, place in 37 C incubator overnight.

Day two:

Make and autoclave 150 ml LB without antibiotic again except for pLys.
Make TSS (18 ml Is more than enough for 100 ml prep)

10 % Polyethylene glycol 3350 2.0 g

5 % Dimethyl Sulfoxide (DMSO) 1 ml

20 mM MgCl₂ (1 M stock) 4 ml

Add LB to 18 ml

Filter sterilize through 0.22 um syringe filter

Chill until needed

Inoculate room temperature LB with cells from a single colony. Grow cells until OD (600) reaches approx 0.4-0.5. (This time varies greatly with the cell strain.)
After OD reaches 0.4, take cells out and put them on ice IMMEDIATELY

(Everything must be kept cold from this point on. Chill pipets, tips, centrifuge tubes, final storage eppi tubes..)

Put cells in the chilled centrifuge tubes and spin down, use big rotor at 6000 g, 4 C for 10 min.
Decant off liquid from cell pellet. (pellet will be small). Place pellet back on ice.
Resuspend in 10 % initial volume of TSS. (i.e. 100 ml cell culture, resuspend pellet in 10 ml TSS)
Aliquot cells into pre-labeled, pre-chilled eppi tubes with pre-chilled pipets. Make aliquots from 30 ul to 100 ul.

Centrifuge Bottle Limits

500ml PPCO 13,700 X g

250ml PPCO 27,500 X g

50ml PPCO 50,000 X g

16ml PPCO 50,000 X g

1 M Dithiothreitol (DTT)

Stock 32.4ml

DTT FW 154.25 5 g

Filter Sterilize with 0.22um syringe filter.

Aliquot into 1 ml. Store at –20 C

Fisher BP172-5

Destain I

1 L

10 % HOAc 100ml

40 % MeOH 400ml

ddH2O 500ml

Destain II

1 L

5 % MeOH 50ml

7 % HOAc 70ml

dd H2O 880 ml

Dialysis Buffer 195

Stock 2 L

25 mM NaPO₄ 1M 50 ml

25 mM NaCl FW 58.44 2.92g

10 mM BME 14.4 M 0.714ml x 2

De-gas by vacuum filtration.

Ethidium Bromide 1000 X (0.5 ug/ml)

Dissolve 5 mg Et. Br in 10 ml ddH₂O

Or 50 mg Et. Br. In 100 ml dd H₂O

Use amber bottle

Keep at room temperature

Working solution : 0.5 ug/ml

e.g. for 100ml gel, use 100.0 ul Et. Br soln.

Gel Drying Solution/ Gel drying directions

40 % EtOH 400 ml

5 % Glycerol 5 ml

Soak gels in soln. above for approx. 30 min
Soak Cellophane in soln. until completely wet.
Place gels between two sheets of cellophane and push out bubbles.

LB Agar Plates

100 ml (~ 6 plates)

Tryptone 1 g

Yeast Extract 0.5 g

NaCl 1 g

Agar 1.5g

Fill to 100 ml final volume with ddH₂O.

Solutes will dissolve when autoclaved

LB Miller

25ml 100ml 500ml 1L

Tryptone 2.5 g 1 g 5 g 10 g

Yeast Extract 1.25g 0.5 g 2.5 g 5 g

NaCl 2.5 g 1 g 5 g 10 g

NaOH (1 M) 0.25 ml 0.1 ml 0.5 ml 1 ml

Agarose Gel Electrophoesis

% Agarose Size DNA Separated

0.3 5 – 60 kb

0.5 1 – 30

0.7 0.8 – 12

1.0 0.5 – 10

1.2 0.4 – 7

1.5 0.2 – 3

2.0 0.05 – 2

Small Gel:

40 ml TAE

40 ul Et Bromide (0.5 mg/ml)

250 ml Buffer

250 ul Et Bromide

Votage: 1 – 5 V/cm (cm between electrode) ~ 55V

Detection limit: ng scale

Glycerol Stocks

1. Inoculate a single colony into 50 ml media w/ amp in a 250 ml flask.

2. Incubate with vigorous shaking at 37 C until OD 600 = 0.6 to 0.8.

3. 0.9 ml cells

0.1 ml 80% glycerol

4. Mix well, store at –70 C

6 M HCl

120 ml H₂O

129 ml of Conc. HCL , 11.6 M (from under hood)

40% Glucose (2.2 M Glucose)

Glucose 40 g

ddH₂O fill to 100 ml

Dissolve in about 50 ml warm H₂O with stirring

Then add the remaining H₂O

Also called Dextrose, FW 180.16

Warm gently to dissolve. Filter sterilize through 0.22 um

Flame top with each use

0.1M HCl

Used in wash bottle for RNA work.

Volume of wash bottle 750 ml.

Add 6.5 ml of conc. HCl, then fill to volume with ddH₂O

1M IPTG

IPTG 2.38 g

Fill to 10 ml with ddH₂O

IPTG 3 g

Fill to 12.6 ml final volume

Filter sterilize with 0.22 um syringe filter.

Aliquot in 1 ml, store at –20 C.

Inclusion Bodies

Three things to do to avoid formation of inclusion bodies

1. Change Temperature. Grow at 30 C instead of the usual 37 C

2. Change cell line

Try BL21(DE3)

HMS174(DE3)

3. Lower level of expression.

Kanamycin 1000x

Kanamycin 0.9 g

Fill to 30 ml final volume with ddH₂O

Filter sterilize, aliquot to 1 ml

Makes a 30 g/L stock

keep at –20 C

use at 1000-fold dilution – 30 ug/L

1M KCl

KCl 7.46 g

Fill to 100 ml final volume with ddH₂O

No need to sterilize.

10 X Loading Buffer: For Agarose Gel

Stock 5 ml

20 % Ficoll 400 (w/v) 1 gm

0.1 M Na₂EDTA pH 8.0 0.5 M 1 ml

1.0 % SDS 10 % 0.5 ml

0.25 % (w/v) BPB 12 mg

Until color blue fades

Lysis Buffer (for NC p7 prep)

50 mM Tris-HCl pH 8.0

10% Glycerol

0.1 M NaCl

0.1 mM ZnCl₂

Add

DTT to 5 mM (for 200 ml add 1 ml of 1M DTT)

EDTA to 2 mM ( “ “ 0.8 ml of 1 M EDTA)

Molar Extinction Coefficient (ε)

n To estimate ε280 of proteins based on sequence.

No. of Tyr x 1280

No. of Trp x 5690

No. of Cys x 120

Add three numbers = ε m-1cm-1

Ref: S.C. Gill & P.H. von Hippel

Anal. Biochem. 182, 319-326, 1989

Calculate Molecular Weight of Oligos

MW= A x 312.2

G x 328.2

C x 288.2

+T x 303.2

Then subtract 61.0

yields concentration in g/mol

M9 Minimal Media (Maniatis)

M9 Salts 1L

Na₂HPO₄·7H₂O 12.8 g

KH₂PO₄ 3 g

NaCl 0.5 g

NH₄Cl 1.0 g

1M MgSO₄ 2 ml

Glucose 4 g

1 M CaCl₂ 0.1 ml

For some proteins (e.g. Zinc finger Proteins)

1 M ZnCl₂ 0.1 ml

100X Vitamin mix 10 ml (Gibco Cat # 11120-052)

1000X Trace metal 1.0 ml (Recipe below)

2M MgCl₂

MgCl₂·6H₂O 40.66 g

Fill to final vol. of 100ml

Autoclave to sterilize, flame top each use

M9TB & 0.4 % Glucose

TB 1 L

H₂O 900 ml

Tryptone 12 g

Yeast Extract 24 g

Glycerol 4 ml

Autoclave

M9ZB Media

1 L

Tryptone 10 g

NaCl 5 g

Autoclave, cool. Then add:

10X M9 salt (see below) 100 ml

IM MgSO4·7H2O 1 ml

40 % glucose* 10 ml*

*Don’t add for overnight cultures.

10X M9 Salt

1 L

NH₄Cl 10 g

KH₂PO₄ 30 g

Na₂HPO₄·7H₂O 60 g

Autoclave.

Use in making M9ZB Media.

Non-Denaturing Gel Buffers

Separating Gel Buffer:

90.85 g Tris-Base

Fill with DI H₂O to 500 ml

Stacking Buffer:

15.43 g Tris-Base

Fill with DI H2O to 500 ml

Potassium Phosphate & M9

100 ml

KH₂PO₄ 2.31 g + 3 g

K₂HPO₄ 12.54 g

NH₄Cl 1.00 g

Na₂HPO₄·7H₂O 6.00 g

Autoclave.

NaOH 0.1 M for Wash Bottle used in RNA work

Wash bottle volume 750 ml

Add 5.3 g NaOH, fill bottle to volume

NMR Buffer for NC 55

Stock 30 ml 40 ml

25 mM d₃-acetate FW 85.06 0.06457 g 0.086 g

25 mM NaCl 2 M 0.375 ml 0.50 ml

0.1 mM ZnCl₂ 0.2 M 0.015 ml 0.20 ml

0.1 mM BME 14.4 M 2 ul 0.003 ml

Use 50 ml Falcon Tube.

Degass by Argon, 15 min

Filter Sterilize using 0.22 um filter

Exchange in Amicon 4 – 5 times.

Extinction Coefficient for DNA (ε)

A x 15.4 ml/umol

G x 11.7 ml/umol

C x 7.3 ml/umol

T x 8.8 ml/umol

Add up all values = ε

Concentration = OD 269 = umol

ε ml

Pepstatin A (1 mg/ml) or 1000 x

ICN cat no. 195368, 10 mg

Dissolve 10 mg in 10 ml freezer cold EtOH

Store at –20 C.

Use at 1 ug per ml cell lysate

Ex. 30 ml Lysis buffer = Add 30 ul Pepstatin A solution

100 ml Lysis buffer = Add 100 ul

10 % PEI

20 ml 50 % (w/v) stock J. T. Baker PEI and H2O

Add conc. HCl to adjust pH to 7.9 or 8.0.

Filter through 0.45 um filter

Store at 4 C

PMSF

10 mM Use at Cell Lysis

575 λ 100 ml

Make stock in Isopropanol

Phenol/Chloroform Extract

Add 1 Vol phenol/chloroform/isoamyl alc. Vortex 10 sec.
Spin, Room temp, 2 min. Transfer top layer to another tube.
Add 1/10 vol 3 M Sodium Acetate, pH 5.2
Add 2.5 vol ice cold 100 % EtOH. Vortex.
Spin down, 5 min, 4 C, Decant, Let stand.

(more?…)

Protease Inhibitors

Use At Stock

Leupeptin 1 –2 ug/ml 10 mg/ml, H2O

Pepstatin A 1 ug/ml 1 mg/ml, EtOH

PMSF 100 ug/ml 1.74 mg/ml, 10 mM IPA

EDTA 1 mM 0.5 M, H₂O

RNA Analytical (Denaturing) Gels

1. Wipe off inside of small plates, small spacers and comb with ethanol.

2. Grease the square plate, approx ¼” in from edge on three sides. Do not get grease in the middle of plate. Place spacers on grease on plate.

3. Mix 20 % denaturing gel solution according to the recipe for 45 ml (below)

4. Slowly pour gel onto bottom plate while sliding up top plate with outher hand. Be careful not to cause bubbles while pouring.

5. Be sure comb is in position so that top of wells are visible above top of notched plate.

6. Clamp around all sides, including around comb.

7. Allow gel to polymerize for 1 hr. (some suggest 1.5 hr is best)

8. Make 1 L 1X degassed TBE buffer.

9. Secure gel with clamps onto electrophoresis apparatus. (Do not remove comb yet)

10. Add 1+/- L of TBE buffer so that bottom of gel is covered and fill top so that top of wells are covered.

11. Remove comb carefully, trying not to lose any wells. Try running a razor blade along each side of comb before removing.

12. Add 2 ul blue-green dye and begin 1 hr- 1.5 hr prerun. Most RNA settings are 900 Volts, 300 mA, 3-7 Watts.

13. Load samples, which are all in 25% glycerol.

14. Add another 2 ul of blue-green dye.

20 % RNA, Denaturing, Purification Gels

Big Gels Analytical Gels

Sequa-gel Concentrate 400 ml 24 ml

Sequa-gel Diluent 50 ml 3 ml

Sequa-gel Buffer 50 ml 3 ml

10% APS 3.5 ml 210 ul

TEMED 170 ul 12 ul

Total Vol 500 ml 30 ml

12% RNA, Denaturing, Purification gels

Beginner Expert

Sequa-gel Concentrate 240 ml 216 ml

Sequa-gel Diluent 210 ml 189 ml

Sequa-gel Buffer 50 ml 45 ml

10% APS 3.5 ml 3.15 ml

TEMED 170 ul 153 ul

RNA Gel Running Protocol

1. Wear gloves and face shield at all times.

2. Wipe off inside of clean gel plates with ethanol.

3. Grease sides and bottom of the bottom plate (the plate that is rectangular) with vacuum grease. Approx. 1/4 inch in from edge. Avoid getting grease elsewhere on plate.

4. Wipe off spacers (two long, one short) and comb with ethanol and place spacers on the grease on bottom plate. (Set comb aside)

5. Mix gel solution following recipe for desired percentage of acrylamide.

6. Slowly pour gel onto bottom plate while sliding up top plate with other hand. Be extra cautious not to cause bubbles while pouring. (bubbles, however, can be removed by using a clean analytical gel spacer).

7. Be sure both plates are squarely together and the spacers seal together the corners to avoid leaks.

8. Clamp around all sides and at the comb.

9. Allow to polymerize for 1 hr.

10. Make about 1.5 L 1X degassed TBE buffer per gel.

11. Once gel has solidified, wipe off outside notched plate. This is very important as not to ruin the electrophoresis equipment. Wipe off gold plate on apparatus as well.

12. Secure gel, notched side to gold plate, into apparatus.

13. Tighten opposing knobs simultaneously using an equal amount of torque on each knob. (This will help avoid breaking plates and plastic knobs.)

14. Be sure drain in back of apparatus is closed and fill top and bottom of apparatus with 1 X TBE buffer.

15. Use bent needle to remove air bubbles from bottom of gel, between plates.

16. Prerun gels for approx. 2 hrs at (usually) 60-80 watts, 300 mA, 900 Volts.

17. Add 2 ul of blue-green dye when pre-running, as a marker, into each of the two small wells on the far sides of the gel.

16. During this time, clean up any spilled gel as it contains acrylamide which is very toxic (and messy).

18. Load sample. No more than 2 ml should be loaded in each well. The optimum amount depends on the depth of each particular well and the experience of the loader.

19. Load another 2 ul of blue-green dye.

20. Run sample on gel at a rate and time pre-determined on the size of the molecule being purified and time availability.

RNase A

5 mg RNase A (BMC),

5 ml TE, pH 8.0

Boil 15 min

Aliquot into 0.5ml.

Store at –20 C

2X SDS Gel Loading Buffer

Stock 50ml

100 mM Tris HCl, pH 6.8 1 M 5 ml

200 mM BME 14.4 M 700 ul

4 % SDS 10 % 20 ml

0.2 % Bromophenol Blue Solid 0.1 g

20 % Glycerol 100 % 10 ml

SDS Loading Dye

Stock 10 ml

Tris HCl pH 6.8 1 M 1.3 ml

SDS 10 % 5.3 ml

Bromophenol Blue Solid ~mg ( a pinch. Very scientific)

Glycerol 100 % 2.6 ml

Store at –20 C in 1 ml aliquots

Use 1000x of dye with 18 λ BME

SDS-PAGE (2x) Sample Buffer

Stock 10 ml

Tris HCl 0.5 M 2.5 ml

Glycerol 100 % 2.0 ml

SDS 10 % 4.0 ml

Bromophenol Blue 0.1 % 0.5 ml

Use at least one part sample buffer to one part sample.

Silver Stain (ICN Kit)

Wash Gel in H₂O 15 min
Pretreatment of solution 10 min
Silver Stain Solution 25 min
Wash 3x with H₂O, 2 min
Develop
Pour off developer
Stop in 3x H₂O wash

Pretreatment soln. Stain Soln. Developer

45 ml H₂O 5 ml (2) 95 ml H₂O

40 ml MeOH 5 ml (3) 2.5 ml (5)

10 ml EtOH 5 ml (4) 2.5 ml (6)

5 ml Soln 1 85 ml H₂O

Stains-All (Sigma E9379)

Stains-all 1.5 g

Formamide 600 ml

H₂O 400 ml

Keep at 4 C. Can be reused.

Stain gel 20 min.

Rinse gel with H₂O.

Do Not Stain Ag Stained gel w/ stains-all. (will destroy stains-all soln.)

SOB/SOC Media

SOB: 1 L 500ml 200ml

Tryptone 20 g 10 g 4 g

Yeast Extract 5 g 2.5 g 1.25 g

NaCl 0.5 g 0.25 g 0.10 g

1 M KCl 2.5 ml 1.25 ml 0.5 ml

1 M NaOH 1 ml 0.5 ml 0.2 ml

(autoclave above ingredients)

Add Before use:

2 M MgCl₂ 5 ml 2.5 ml 1 ml

For SOC add:

40 % Glucose 9 ml 4.5 ml 1.8 ml

Siliconizing Solution (RNA Plates)

500 ml

20 % v/v Dichlorodimethylsilane 100 ml

80 % v/v Methylene Chloride 400 ml

Sodium Azide (NaN₃) (poison!!)

Use 0.01 % for NMR samples

Use 0.05 % for G sephadex

---(?)----

Trace Metals

1% FeCl₃ . 6H₂O 2.45 ml

1% NaMoO₄ . 2H₂O 0.01 ml

1% ZnSO₄ . 7H₂O 2.0 ml

1% MnSO₄ . H₂O 0.085 ml

1% H₃BO₄ 0.075 ml

1% CoCl₂ . 6H₂O 0.25 ml

1% NiCl₂ . 6H₂O 0.05 ml

H₂O 45.08 ml

Tris-Glycine Electrophoresis Buffer (10X Laemmli Buffer)

10 X stock 500 ml 1 L

1.92 M Glycine 72.1 g 144.2 g

0.25 M Tris 15.1 g 30.2 g

1.0% SDS 5.0 g 10.0 g

Dissolve in warm water.

Good for 6 months.

Tris-Tricine Running Buffer

10X, 500 ml 10X, 300 ml

Tris Base 60.58 g 36.35 g

Tricine 89.6 g 53.76 g

SDS 5.0 g 3 g

Run at 100 V, 150 min. Blue dye to reach end.

Tris-Tricine Gel Stain

500 ml : 50 ml HOAc

0.125 g G-250 Coomassie

To Stain:

1. Fix gel 30 min in Destain I

2. Stain 1 hour in Tris-Tricine stain solution.

3. Destain 3X 15 min, 10% HOAc

4. Keep in Destain II overnight

TB

A) Tryptone 12 g

Yeast Extract 24 g

Glycerol 4 ml

H₂O 900 ml

B) KH2PO4 2.3 g

Yeast Extract 12.5 g

H₂O 100 ml

Autoclave Separately.

When at room temperature, add and mix together.

10 ml B to 90 ml A

TSS (for Competent Cells)

Stock Add

LB LB 4.5 ml

10 % PEG 3350 (w/v) Solid 0.5 ml

5 % DMSO Liq. 0.250 ml

20 mM MgCl₂ I M 0.100 ml

Use 15 ml Falcon tube.

No need to pH, it should be around 6.4-6.8.

Filter sterilize through 0.22 um filther

Place on ice until added to cells

If stored for later, store at –20C

TE , pH 7.4, pH 7.5, pH 8.0

Stock 100 ml (10x) 10 ml

10 mM Tris-HCl 1 M 1 ml 10 ml 0.100 ml

1 mM EDTA 0.5 M 0.2 ml 2 ml 0.020 ml

Sterilize.

TAE Electrophoresis Buffer 10X stock

1 L 500 ml

Tris Base 48.4 g 24.2 g

Glacial Acetic Acid 11.42 ml 5.71 ml

EDTA free acid, FW 292.2 5.84 g 2.92 g

1X working Concentration:

40 mM Tris acetate

2 mM EDTA

10X TBE (not for RNA)

1 L 500 ml 250 ml

Tris base 108 g 54 g 27 g

Boric Acid 55 g 27.5 g 13.8 g

EDTA, 0.5 M, pH 8.0 40 ml 20 ml 10 ml

1X working Concentration:

89 mM Tris

89 mM Boric Acid

2 mM EDTA

Tetracyline Stock

10 ml (H2O)

Tetracyline 0.2 g

Store at –20 C.

Trace Metals Mix (2000 X)

Use at 0.5 ml per liter prep

FW Metal metal/liter Add per liter 40 ml

FeCl₃·6H₂O 270.0 55.85 10 ug/L 48.4 x 10^-60.48

CuSO₄·2H₂O 170.5 63.5 10 ug/L 26.9 0.27

MnCl₂·4H₂O 197/9 54.9 10 ug/L 36.1 0.36

ZnCl₂ 136.28 65.4 4 mg/L 1.67 g

CoCl₂·6H₂O 237.9 58.9 2 ug/L 8.08 0.081

Na₂MoO₄·2H₂O 241.9 95.9 2 ug/L 5.04 0.050

NiCl₂·6H₂O 237.7 58.9 2 ug/L 8.07 0.081

Vit Mix

Storage 40 ml 1 L prep working concentration

Thiamine R.T. 0.1 g 3 mg

Choline R.T. 0.1 g 3 mg

Niacinamide R.T. 0.1 g 3 mg

D-Pantothenate 4 C 0.1 g 3 mg

Pyridoxal -20 C 0.1 g 3 mg

Folic Acid R.T. 0.1 g 3 mg

Filter sterilize 0.22 um filter

Aliqout into 5 ml. Store at –20C.

Use at 0.5 ml per 1 L prep.

ZB Top Agar

1 L