Solutions, Media and Other Preparations

stdpreps.doc 10/13/18 Page 1

Standard Preparations

Solutions, Media and Other Preparations

Acetylated BSA

This is supplied commercially (e.g., Promega supplies 1 mg/ml)

Acrylamide/TBE gels (5–15%, for DNA and RNA)

This is the recipe for 45 ml (for Hoefer "Sturdier" vertical electrophoresis apparatus); reduce by a factor of 5 for minigel apparatus. We use acrylamide concentrations between 5% and 15%.

required amount of 30% stock (29% acrylamide/1% bis w/w) (7.5–22.5 ml)

9 ml 5 × TBE

900 l 3% w/w ammonium persulfate (freshly dissolved in water)

water as necessary to bring total volume to 45 ml (add water first)

degas in vacuum flask

Add 27 l TEMED, pour between plates, put in well-forming comb. Allow to polymerize ~1 hr.

AP-SA (0.5 mg/ml stock of alkaline phosphatase-conjugated streptavidin)

We obtain vials with 1 mg of conjugate from Jackson ImmunoResearch, and reconstitute as outlined in the following steps. This procedure assumes the conjugate is supplied as a 1-mg/ml solution in buffer. If instead the conjugate is supplied as a lyophilized powder, dissolve the powder in 1 ml of water before adding the glycerol/salt solution at step b.

a. In a 1.5-ml Ep tube measure:

30 µl 1 M MgCl2
3 µl 1 M ZnCl2
1.89 g (1.5 ml) ultrapure glycerol

Add the two salts first, then weigh in the glycerol; close cap and mix thoroughly by vigorous vortexing and repeated inversion. Put tube on ice to get it ice-cold.

NOTE: In the following steps, keep the vial of alkaline-phosphatase-conjugated streptavidin ice-cold as much as possible. Return the vial to ice after each manipulation to keep the solution cold.

b. Centrifuge the commercial vial from Jackson Labs briefly in clinical centrifuge. Open the vial, keeping the rubber septum clean. Tare the vial, then add 1.26 g (1 ml) of the above ice-cold mixture. Close the rubber septum, wrap the top of the vial with parafilm. Tape the vial to a rotater and rotate for ~30 min in the cold to thoroughly mix the contents.

c. Centrifuge the vial briefly in the clinical centrifuge to drive solution to bottom. Open septum and divide the solution equally between two 1.5-ml Ep tubes. Store in the refrigerator.

AP-SA diluent

50 mM 1 M Tris-HCl pH 7.5

150 mM NaCl

0.1% Tween 20

1 mg/ml dialyzed BSA

ATP (100 mM stock)

Weigh out 50–100 mg ATP disodium salt (F.W. 605.2) into a sterile 4-ml tube

Note exact weight; for each mg of ATP add:

13.8 l water

2.56 l 2 M Tris (pH unadjusted; ~3 mol Tris/mol ATP)

Spot-test pH: if not between 7 and 9, adjust pH with 1 N NaOH or HCl

Scan a 1/4000 dilution from 220–300 nm; should peak at A259 ~0.4

Determine concentration in undiluted stock

assuming molar absorbance coefficient of 15,400 at 259 nm

should be ~100 mM

Store at –20º

Biotin (10 mM stock)

Make a 10 mM solution in water

With constant stirring, add 1 M NaOH dropwise

Blocking solution

0.1 M NaHCO3

5 mg/ml dialyzed BSA

0.1 g/ml streptavidin

0.02% NaN3

Make 150 ml, filter-sterilize and store in refrigerator; we keep re-using the solution until it shows evidence of microbial growth or accumulation of insoluble matter.

BSA (ordinary)

As a carrier for nucleic acid enzymes, use acetylated BSA; as a carrier for components to be exposed to streptavidin, use dialyzed BSA. Ordinary BSA is fraction V protein (Sigma A-2153). It is made up as a 50 mg/ml solution in autoclaved water, filter-sterilized, and stored in the refrigerator.

Buffered glucose

50 mM glucose

25 mM Tris.HCl pH 8

10 mM EDTA

Autoclaving or filter sterilization is optional; store in refrigerator

CaCl2 (0.1 M stock)

Autoclave in 125-ml bottles

Store in refrigerator if it will be used for making competent cells (CalciumChlorideTransfection.doc)

Chloroform (with isoamyl alcohol)

Sterilely add 20.8 ml isoamyl alcohol to a new 500-ml bottle of chloroform. Both solvents are reserved for DNA work to preserve sterility (not only microbiological, but enzymological).

Dialyzed BSA (hopefully biotin-free)

Protein is from Sigma (Cat. # A-3912)

Dissolve at 50 mg/ml in water

Filter-sterilize

Store at –20º

Diethanolamine pH 9.8 (1 M stock for ELISA)

Adjust pH with HCl

Need not be autoclaved

DNaseI (2258 units/ml stock)

Dissolve contents of 1 vial of Worthington DPRF (11,290 Kunitz units/vial; 5.6 mg total dry weight/vial, much of which is glycine and calcium stabilizers) in 5 ml autoclaved, ice-cold 50% v/v glycerol

Store at –20º.

Double-spin method

NOTE: This method greatly increases the yield of aqueous phase from each organic extraction. The procedure given is for small volumes in Ep tubes; use larger centrifuge tubes (phenol and chloroform resistant!!) and pipetting devices as necessary for larger volumes.

1. To ~500 l aqueous phase in a 1.5-ml Ep tube (or 200 l in a 500-l Ep tube) add equal vol of the organic phase (phenol, chloroform, or phenol/chloroform).

2. Vortex vigorously

3. Microfuge to separate phases

4. Using a 200-l pipetter with a yellow tip, carefully draw off the organic (lower) phase, leaving all the interphase and upper phase behind. The idea is to lower the interphase into the narrow tip of the tube so the aqueous phase can be drawn off with high yield. Try to avoid removing upper phase.

5. Re-microfuge to re-separate phases (the “second spin”).

6. Remove the upper (aqueous) phase with a yellow pipette tip, being very careful to avoid any interphase or lower phase. Usually the aqueous phase will be transferred to another Ep tube for extraction or ethanol precipitation.

D-PBS (Dulbecco’s PBS)

10 × stock (1 liter in plastic bottle)

26.7 mM KCl

11.5 mM KH2PO4

1.379 M NaCl

80.6 mM Na2HPO4

pH should be between 6.7 and 6.9

If not, adjust to 6.8 with 1 N HCl or 2 N NaOH

store in refrigerator

1 × solution

In a 1-liter beaker mix 100 ml 10 × stock and 900 ml water

Check that pH is between 7.0 and 7.2

If not, adjust to 7.1 with 1 N HCl or 2 N NaOH

Dispense 200 ml into each of five wide-mouth 250-ml polypropylene bottles

Autoclave

When cool, to each 200-ml portion add:

2 ml 0.1 M CaCl2 (final conc. 1 mM)

100 l 1 M MgCl2 (final conc. 0.5 mM)

Store at room temperature

DTE (0.5 M dithioerythritol; dithiothreitol is equivalent)

Dissolve 0.77 g in 9.23 ml sterile water, trying to minimize contamination

Filter-sterilize into a 15-ml bottle

Store at –20º

EDTA (250 mM stock)

250 mM Na2EDTA

Adjust pH to 8.0 with NaOH

Autoclave

Store at room temperature

Elution buffer

0.1 N HCl, pH adjusted to 2.2 with glycine

1 mg/ml BSA

0.1 mg/ml phenol red is optional

The glycine.HCl buffer is made and adjusted as a 4 × stock, filter-sterilized, and stored at room temperature; this buffer and the other components are made up in autoclaved water and the whole solution is filter-sterilized and stored in the refrigerator. The phenol red gives a visual indication of whether the pH is (roughly) correct after the eluate is neutralized.

Ethanol (70% v/v; 64.9% w/w)

Autoclave ~39 ml water in a tared 125-ml bottle (record tare weight)

Determine net weight of water after autoclaving

Add 1.85 grams 100% ethanol for every net gram of water

Store at –20º

Formamide load buffer

6.65 ml formamide (ultrapure)

350 l 250 mM EDTA

3.5 mg bromophenol blue (dark blue tracking dye)

3.5 mg xylene cyanol FF (blue-green tracking dye)

Store in 10-ml amber bottle in deepfreeze or refrigerator

GBB (40 × stock)

142.4 g Tris

45.94 g anhydrous sodium acetate (or 76.16 g trihydrate)

18.83 g Na2EDTA·2H2O

Dissolve in ~500 ml water

Adjust pH to 8.3 with glacial acetic acid

Adjust final volume to 700 ml

Store nonsterilely at room temperature

HEPES pH 7 (100 mM stock)

Adjust pH with NaOH

Autoclave

Store at room temperature

Hoechst dye (1 mg/ml stock)

Dissolve Hoechst dye 33258 in water at 1 mg/ml

Store in an amber bottle in refrigerator

IPTG (0.2 M stock)

Make 10 ml

Filter-sterilize into a sterile 15-ml bottle

Store at 4º

Isocitrate (pH 7.1; 1 M stock)

1 M D,L-isocitrate, trisodium salt

Adjust pH to 7–8 with HCl (~10 mmol HCl required/mol isocitrate)

Autoclave

Store at –20º

Kanamycin (100 mg/ml stock)

Dissolve kanamycin sulfate to 80 mg/ml in water

Adjust pH to 6–8 if necessary with NaOH or HCl

Filter sterilize

Store at 4º

KCl (1 M stock)

Autoclave

Store at room temperature

Kinase buffer (10 × stock)

0.5 M Tris.HCl pH 7.5

100 mM MgCl2

50 mM DTE

1 mM spermidine

1 mM EDTA

Make from sterile water and stock solutions

Store at 20

KOAc (“5 M” stock; actually 3 M KOAc, 2 M HOAc)

29.44 g potassium acetate (anhydrous)

71.68 g water

12.06 g glacial acetic acid

Store in refrigerator; don't autoclave

LB (1 ×)

10 g/liter bacto-tryptone

10 g/liter NaCl

5 g/liter yeast extract

Adjust pH to 7.0 with NaOH

Autoclave, store at room temperature

For plates make 2 × stock, autoclave and store at room temperature

Ligation buffer (5 × stock)

Pipette stock solutions into sterile 500-l Ep tube to give following final concentrations:

150 mM Tris.HCl pH 7.5

150 mM NaCl

37 mM MgCl2

10 mM DTE

1 mM EDTA

5 mM spermidine

0.5–1 mg/ml acetylated BSA

1.25 mM ATP

Store at –20º

Lysis mix

2 g SDS

18 ml water

2 ml 40 × GBB

40 mg bromphenol blue

20 ml glycerol

USE: Add 1 vol to 4 vol of electrophoresis sample

MgCl2 (1 M stock)

Autoclave

Store at room temperature

MgSO4 (0.4 M stock)

Autoclave

Store at room temperature

MnCl2 (50 mM stock)

Autoclave;

Store at room temperature

NaCl (5 M stock)

29.22 g NaCl plus 88.78 ml water makes 100 ml (density 1.18 g/ml)

Autoclave in glass bottle with cap tight to preserve concentration

Store at room temperature

NaCl (80 mM, for preparation of concentrated cells)

Make 1 liter in a polypropylene bottle by diluting 5 M NaCl

Autoclave; store at room temperature)

NaN3 (5% stock)

DANGER: sodium azide is toxic. Handle with gloves, guard against spills

Dissolve 0.5 g solid in 9.5 ml water (don’t autoclave)

Put in 15-ml bottle

Store in refrigerator, with appropriate warning on tube

NaOAc (3 M stock, pH 6)

3 M sodium acetate

adjust pH to 6 with glacial acetic acid

autoclave in a tightly-stoppered screw-cap bottle to prevent evaporation of HOAc

store at room temperature

NAP buffer

Make 150 ml of

80 mM NaCl

50 mM NH4H2PO4 pH 7.0 with NH4OH

The ammonium phosphate buffer is made as a 0.5 M stock and autoclaved with cap on tight to prevent evaporation of the volatile ammonia

Filter-sterilize

Store in refrigerator

NENSORB reagent A

Make 0.1 M Tris, 1 mM Na2EDTA

Adjust pH to 7.7

Autoclave

Add 14 l reagent-grade triethylamine per 10 ml buffer

Store in refrigerator

Neutralizer (for sequencing reactions

0.173 N HCl (made from commercial titrated 1 N HCl)

192 mM Tris.HCl pH 7.2

34 mM D,L-isocitric acid (pH adjusted to 7.2 with NaOH)

11.3 mM MnCl2

100 µg/ml (282 µM) phenol red

Make a 1.023 × stock solution without MnCl2 and store at room temperature

Just before use, mix 1 vol 0.5 M MnCl2 with 43.25 vol stock

Volume required: 6 µl per template plus extra for pipetting errors

NH4OAc (7.5 M stock)

Dissolve 53 g ammonium acetate in 47 ml water

Autoclave in tightly stoppered screw-cap bottle to prevent evaporation of the volatile salt

Store at room temperature

NPP substrate (50 mg/ml p-nitrophenylphosphate in water)

Dissolve 500 mg in 10 ml water

Dispense 100-µl aliquots into 96 minitubes

Store the minitubes at –20º; thaw individual aliquots as necessary

NOTE: We don’t cover the individual minitubes, but we do keep the minitubes in a covered rack in order to minimize sublimation.

NZY medium (1 ×)

NOTE: This is our routine rich medium; other media, such as LB, can also be used. NZY has the advantage that NZ amine A (from Humko Sheffield Chemical, P.O. Box 630, Norwich, NY 13815) is much cheaper than tryptone.

10 g NZ amine A

5 g Yeast extract

5 g NaCl

Dissolve in 1 liter water

Adjust pH to 7.5 with NaOH

Autoclave

Store at room temperature

For making plates, make 2× stock, store at room temperature

PCR buffer (10 ×)

0.5 M KCl

15 mM MgCl2

100 mM Tris.HCl pH 8.3

PEG/NaCl (16.7%/3.3 M stock)

100 g PEG 8000 (Union Carbide; used to be called PEG 6000)

116.9 g NaCl

475 ml water

stir until solutes dissolve (may be necessary to heat to 65 briefly to dissolve the last crystals of PEG)

Store at 4 (total volume 600 ml)

This solution can be autoclaved with no apparent ill effects; as it cools, shake occasionally to prevent separation into two phases. If this separation occurs, re-autoclave to re-homogenize the solution.

Phenol

Water-saturated stock

To redistilled phenol add an equal volume of water

Liquify by heating to 45-65

Shake to equilibrate the two phases (lower phase is phenol)

Store at 4 away from light

NOTE: This stock phenol is acidic, and is stable for months. It must be neutralized (as below) before use, but neutralized phenol only lasts a few days. Discard phenol at the first sign of a yellowish tinge.

Neutralized phenol

Remove required volume water-saturated phenol (lower phase) to suitable vessel

Add 1/10 volume 1 M Tris.HCl pH 8–8.5

Shake or vortex vigorously to equilibrate phases

Separate phases by centrifugation

(or by gravity in separatory funnel for large volumes)

Discard the upper (aqueous) phase in hazardous waste

Equilibrate with buffer once more, again discarding upper phase

Use the lower phase as neutralized phenol

This solution can be stored a few days at 4 away from light

Phenol/chloroform

In a suitable vessel, shake or vortex vigorously:

1 vol water-saturated phenol (see above)

1 vol chloroform

1/10 vol 1 M Tris.HCl pH 8–8.5

Separate phases by centrifugation

(or by gravity in a separatory funnel for large volumes)

Discard the upper (aqueous) phase in hazardous waste

Equilibrate with buffer once more, again discarding upper phase

Use the lower phase as phenol/chloroform

Plates

This is the recipe for 1 liter, enough for ~40 plates; scale up or down as appropriate. Two-fold concentrated (2×) liquid medium is made and autoclaved in advance, and stored at room temperature in polypropylene bottles (it’s much easier to pour sterilely out of polypropylene than out of glass, because of the tendancy toward “drip-back” in the latter).

In a 2-liter polypropylene erlenmeyer flask (at least twice the capacity of the final volume; here especially plastic is preferable to glass) measure 500 ml water and 11 grams Bacto agar. Cover with a polypropylene or glass beaker and autoclave.

While the agar is autoclaving, set out and label empty petri dishes

To the autoclaved agar (not cooled) add 500 ml of 2× liquid medium at room temperature. Pour the medium gently down the wall of the flask, which should be held at an angle to prevent the medium from splashing down directly into the agar. These manipulations are designed to minimize bubbles, which are exceedingly hard to remove. Mix the contents of the flask by gentle rotation, holding the flask at a shallow angle to promote mixing; again, avoid bubbles.

The medium is now at the average of room temperature and boiling, i.e. ~60. This is cool enough to add antibiotics and other heat-sensitive supplements. The final medium can be poured immediately or can sit for a while as long as the temperature doesn't fall below 50 (that's just hot enough that you can't keep your hand on the flask indefinitely). If you do allow the medium to sit, don't add heat-sensitive supplements until just before pouring plates.

Pour the medium into the dishes, adding at least enough to cover the bottom.

Allow the plates to cool to room temperature. They should dry overnight at room temperature or a few hours in the 37 incubator before use, as water pools on the surface as the agar sets over a period of a few hours. Plates can be dried within a few hours upside down tilted out of their lids in a sterile laminar flow hood.

REact-1 buffer (10 × stock)

500 mM Tris.HCl pH 8.0

100 mM MgCl2

Autoclave

Store at room temperature

REact-2 buffer (10 × stock)

500 mM Tris.HCl pH 8.0

100 mM MgCl2

500 mM NaCl

Autoclave

Store at room temperature

REact-3 buffer (10 × stock)

500 mM Tris.HCl pH 8.0

100 mM MgCl2

1 M NaCl

Autoclave

Store at room temperature

REact-6 buffer (10 × stock)

50 mM Tris.HCl pH 7.4

60 mM MgCl2

500 mM NaCl

500 mM KCl

Autoclave

Store at room temperature

RnaseA (10 mg/ml; heat-treated)

Dissolve RNaseA at 10 mg/ml in 0.1 M NaCl, 25 mM Na2Citrate pH 5.5

Heat at ~95º for ~3 min

Dispense 200-µl portions into 500-µl Ep tubes

Store at –20º; thaw and refreeze as needed

RRR (remove supernatant from pellet, recentrifuge, remove residual supernatant)

NOTE: We use this procedure when we are trying remove all traces of a supernatant from a centrifugally pelleted precipitate (e.g., PEG-precipitated virions, ethanol-precipitated DNA).

1. After centrifuging or microfuging as appropriate to drive the precipitate to the bottom of the tube, remove the supernatant by decanting (large tubes or bottles), pipetting, or aspirating (microfuge tubes).

2. Re-centrifuge (or re-microfuge) briefly, orienting the tube as in the first centrifugation, to drive residual supernatant to the bottom.

3. Remove residual supernatant by pipetting or aspirating.

SDS (20% w/w stock)

Dissolve 20 g sodium dodecyl sulfate in 80 ml water

Store at room temperature

Serial dilutions

We do serial dilutions on a small scale using pipetters and 2.2-ml capless polypropylene microtubes available from Sarstedt (this company also sells polyethylene tubes of the same size, but they are not autoclavable). Using these small capless tubes dramatically increases the speed and ease of making the dilutions compared to capped tubes, or to tubes that are longer than a pipette tip; however, only ~800 µl can be vortexed in a 2.2-ml tube without danger of spilling over the side. The vortexer speed must be pre-set empirically so as to give good mixing without spilling. The table below gives appropriate volumes of diluent (pre-loaded into the tubes) and concentrate (added from previous tube in the dilution series) for various target dilution factors. If a mixture of dilution factors is to be used in a single series, it is advisable to use the same volume of diluent in all tubes so as to reduce the opportunity for mistakes. For example, we use 700 µl diluent for both 1/10 and 1/100 dilutions: 77.8 µl of concentrate is added to make a 1/10 dilution, 7.1 µl of concentrate to make a 1/100 dilution.

1. Into the appropriate number of sterile, capless 2.2-ml microtubes pipette appropriate amount of diluent (up to ~700 l; see table below).

2. Transfer the appropriate amount (see below) of the undiluted stock or of the previous dilution into the next tube. DISCARD TIP!!! Vortex gently so as to thoroughly mix contents without letting them splash out of the tube (e.g., setting 3–4 on Fisher Vortex Genie 2).

3. Continue the series in the same way.

SOB medium

20 g Bacto-tryptone

5 g yeast extract

0.58 g NaCl

0.19 g KCl

Dissolve in 1 liter high-purity water

Autoclave in 100-ml portions in 125-ml bottles

After cooling, add 1 ml “2 M Mg++” to each bottle

“2 M Mg++” = 1 M MgCl2, 1 M MgSO4, filter sterilized

Store at room temperature

SOC medium

To each 100-ml bottle of SOB add 1 ml 2 M glucose (filter sterilized)

Store at room temperature

Spermidine (500 mM stock)

Dissolve 0.51 g spermidine·3HCl in 3.6 ml sterile water

Filter-sterilize into a sterile 4-ml vial

Store at –20º

Streptomycin (50 mg/ml stock)

Dissolve 0.6 gram streptomycin sulfate in 10.5 ml water

Filter-sterilize into a sterile 15-ml bottle

Store in refrigerator

Usually used at 100 g/ml final concentration

TB soft agar

Into a number of glass 125-ml bottles weigh:

1 g Bacto tryptone

0.5 g NaCl

0.75 g Bacto agar

Add 100 ml water