116102334 02/12/16 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.
116102334 02/12/16 Page 2
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 to pH 7–9
Filter-sterilize into sterile bottle
Store at –20º
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, filtersterilized, and stored in the refrigerator.
Buffered glucose
50 mM glucose
116102334 02/12/16 Page 3
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)
Chloramphenicol (34 mg/ml stock)
 Flame-sterilize a small spatula; use it to weigh 1.7 grams of chloramphicol (Sigma C-0378) into
a sterile disposable50-ml tube.
 Pour in absolute alcohol to the 50-ml mark. Avoid contaminating either the ethanol stock or the
chloramphenicol solution
 Close cap and mix by shaking
 Pour into an autoclaved 60-ml Nalge bottle; store at –20º
Use at 34 µg/ml (1/1000 dilution of the stock) in both plates and liquid medium
3. Label a sterile 60-ml Nalge bottle with blue-green tap “Chloramphenicol 34 mg/ml in ethanol 1/8/06
CAMSTOCK2:3” and label cap with same tape “Chloramphenicol”; pour the stock solution into the 60ml bottle; store in right-hand end of –20°C chest freezer (discard the old stock from CAMSTOCK.doc to
hazardous waste).
USE
Use at 34 µg/ml (1/1000 dilution of the stock) in both plates and liquid medium.
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
116102334 02/12/16 Page 4
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.
116102334 02/12/16 Page 5
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.
116102334 02/12/16 Page 6
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
Glycerol tubes
NOTE: E. coli strains that are used routinely are stored frozen in 25% glycerol as small
aliquots in small one-use tubes such as 500-µl Ep tubes. About 100 prelabeled tubes are
autoclaved along with a bottle of 50% v/v glycerol. When the 50% glycerol is cool, 50µl portions are pipetted into the sterile tubes; meanwhile, a starter culture of the E. coli
strain (at least 6 ml) is shaken overnight; 50-µl portions of the grown culture are pipetted
into the glycerol-containing tubes, which are then vortexed and stored in a –80º freezer.
A single glycerol tube is used to inoculate a starter culture as needed; after this use, the
glycerol tube is discarded, even if some suspension remains in it. This has proven to be a
far more reliable way of inoculating starter cultures than using colonies on plates: we’ve
never been unpleasantly surprised in the morning to see that an overnight culture hasn’t
grown.
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
116102334 02/12/16 Page 7
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 100 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
116102334 02/12/16 Page 8
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)
116102334 02/12/16 Page 9
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
116102334 02/12/16 Page 10
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.
116102334 02/12/16 Page 11
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 or years. 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
116102334 02/12/16 Page 12
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
116102334 02/12/16 Page 13
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
116102334 02/12/16 Page 14
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.
Concentrate volume for indicated dilution
Diluent
1/2
1/3
1/5
1/10
1/100
100 µl
100 µl
50 µl
25 µl
11.1 µl
TS
200 µl
200 µl
100 µl
50 µl
22.2 µl
TS
300 µl
300 µl
150 µl
75 µl
33.3 µl
TS
500 µl
TL
250 µl
125 µl 55.6 µl
5.1 µl
700 µl
TL
TL
TL
77.8 µl
7.1 µl
TL, too large: tube can’t be vortexed with volume > ~800 µl
TS, too small: pipetting errors too high with volumes < ~5 µl
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
116102334 02/12/16 Page 15
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
Autoclave
Store at room temperature
To use for pour plates:
Re-liquify agar in microwave or boiling water bath
Dispense ~3-ml portions into sterile 13 × 100 mm tubes
Put tubes in 50º tempblok to keep the agar liquified
TBE (5 × stock)
Dissolve the following in water to make a total volume of 1 liter
60.5 g Tris
31 g H3BO3
3.7 g Na2EDTA·2H2O
Don’t adjust pH
Store at room temperature
NOTE: We observe a precipitate forming on storage, but continue to use the 5 × stock
without apparent ill effects.
TBS (1 ×)
50 mM Tris.HCl pH 7.5
150 mM NaCl
Autoclave if desired
Store at room temperature
NOTE: we make a 10× stock and store at room temperature without autoclaving
TBS/gelatin
Autoclave 0.1 g gelatin in 100 ml TBS
After autoclaving, swirl to mix in the melted gelatin
Store at room temperature
116102334 02/12/16 Page 16
TBS/Tween
200 vol TBS
1 vol Tween 20 (final conc. 0.5% v/v)
TE (1 ×)
10 mM Tris.HCl
pH adjusted to desired value
(unless otherwise noted, the pH is assumed to be 8.0)
1 mM EDTA
autoclave
store at room temperature
Termination diluent and termination mixes (R, Q, W, M, K and S)
NOTE: The 100 mM dNTP and 5 mM ddNTP stocks referred to below are purchased
from Pharmacia as solutions at near-neutral pH and stored at –20º.
Termination diluent (30 ml in a 30-ml bottle)
Component
Water
1 M Tris.HCl pH 7.5
5 M NaCl
0.5 M DTE
10 mg/ml acetylated BSA
100 mM dATP
100 mM dGTP
100 mM dCTP
100 mM dTTP
Volume
27.92 ml
500 µl
400 µl
800 µl
300 µl
20 µl
20 µl
20 µl
20 µl
Final conc.
16.7 mM
66.7 mM
13.3 mM
100 µg/ml
66.7 µM
66.7 µM
66.7 µM
66.7 µM
Use termination to make termination mixes below; also dispense 1-ml aliquots
into 1.5-ml Ep tubes and store at –20º for making dilutions for sequencing.
116102334 02/12/16 Page 17
Termination Mixes (Q, R, W, M, K and S)
To 10-ml portions of termination diluent add 5-mM (or 0.5-mM) ddNTP stocks as
follows:
Volume to add to 10 ml diluent for indicated mixes:
ddNTP
Q mix
R mix
W mix M mix
K mix
S mix
5 mM ddTTP
6.7 µl
6.7 µl
6.7 µl
5 mM ddATP
6.7 µl
6.7 µl
6.7 µl
6.7 µl
5 mM ddGTP
6.7 µl
6.7 µl
6.7 µl
5 mM ddCTP
6.7 µl
6.7 µl
0.5 mM ddCTP 6.7 µl
6.7 µl
Dispense 1-ml aliquots into 1.5-ml Ep tubes; store at –20º
Terrific broth
In 900 ml water dissolve:
12 g bacto-tryptone
24 g yeast extract
4 ml (5.04 g) glycerol
Autoclave 90-ml portions in 125-ml polypropylene bottles
When cooled, to each bottle add 10 ml of separately autoclaved potassium phosphate
buffer (0.17 M KH2PO4, 0.72 M K2HPO4).
This buffer is made by dissolving 2.31 g KH2PO4 (anhydrous) and 12.54 g
K2HPO4 (anhydrous) in 90 ml water, adjusting volume to 100 ml, and
autoclaving.
Tetracycline (20 mg/ml stock)
In a 100-ml bottle autoclave 40 ml (50.4 g) glycerol
When cool, filter-sterilize 40 ml (= 40 g) 40 mg/ml tetracycline in water into the bottle
Mix thoroughly
Store at 20 (doesn't freeze), away from light
TNE buffer (10 × stock)
100 mM Tris
1 M NaCl
10 mM Na2EDTA
Adjust pH to 7.5 with HCl
Store at room temperature; need not be autoclaved
116102334 02/12/16 Page 18
Tris.HCl buffers (1 M stocks, various pH’s)
Adjust pH to desired level with concentrated HCl at room temperature
Autoclave
Store at room temperature
NOTE: The pH’s quoted in recipes are those of the 1 M stock at room temperature unless
otherwise noted; more dilute solutions have slightly different pH’s, and pH at higher
temperatures can drop a full unit or more.
TTDBA
To TBS/Tween add BSA to 1 mg/ml and NaN3 to 0.02%
X-gal (2% stock solution)
Dissolve in dimethylformamide
Store at 4º in an amber glass bottle with a polyethylene lined cap
NOTE: Dimethylformamide dissolves many plastics, including polystyrene
Yeast RNA (5–10 mg/ml; used as nucleic acid carrier)








Dissolve an entire 100-mg bottle of yeast RNA (Sigma R6750) in 10 ml TE
Extract once with neutralized phenol and once with chloroform/isoamyl alcohol
Divide final aqueous phase evenly into two 15-ml conical screw-cap centrifuge tubes
To each tube add:
o 0.1 vol 3 M NaOAc pH 6
o 2.2 vol 100% ethanol
Vortex; incubate in refrigerator 1 hr.
Centrifuge 12 Krpm 15 min in FiberLite rotor with adaptors for 15-ml tubes for 15 min at
4º; pour off supernatant; recentrifuge briefly to drive residual supernatant to tip, orienting
the tubes as before; aspirate off residual supernatant; air dry briefly (no need to get rid of
last traces of moisture)
Dissolve pellet in each tube in 5 ml TE; pool both solutions into a sterile 15-ml bottle;
store the bottle in the deepfreeze.
Scan a 1/200 dilution from 220 to 320 nm and calculate the RNA concentration,
assuming that a A260 of 1 corresponds to 33 µg DNA per ml; the RNA concentration
turned out to be 7.78 mg/ml
70/75/BPB
70% v/v glycerol
75 mM EDTA
0.3% w/v bromphenol blue
USE: Add 1 vol to 6 vol electrophoresis sample