~
.
Ur ~ Iozm...
.s
.
DIX
APp2
Recipesfor Media,
Reagents,and Stock
Solutions
Solutions
~
Thesuccessof the laboratories in this book dependson the UR of uncontaI
minated reagents.Follow the recipeswith care, and par ICrUpuloUlattmtion to clean1ines..Vse a dean spatula for each ingredient, or cacefully
pour eachingredient from the bouIe.
The recipesare organizedin eigbt sections.Stock solutions that are osed
in more than one laboratory are listed ona; according to their fint Ute.
f
I. BacteriaJCulture
4.. N sodium
sodium hydroxide
hydroxide (NaOH)
(N.OH)
10mg/mi
mg/mI ampiållin
ampicållin
11O
OmgImI
mgIml kanamycin
kanamyån
20
20 mg/mi
mg/mIX-gal
X-gal (S-brom0-4-chloro-3-indoyl-p-D-plactoside)
(S-brom0-4-chlo
Luria
Bertani
(
LB)
Lucia(LB) broth
broth
1.8 brotb + antibiocic
LB agar plata
LB agar + antibiotic
LB agar + antibiotic + X-pl
Stab cultures
II. DNA Restriction
1 M Tris (pH 7.6, 8.0, and 8.3)
5 M sodium ch1oride(N.O)
387
1 M magnesiumchloride (MgCll)
1 M dithiothreitol (DIT)
lOx compromiserestriction buffel
New EnglandBiolabs buffer '1
New EnglandBiolabs buffer 14
30 mM S-adenosylmethionine (S)
(SAM)
2x restriction buffer
0.05% glacial aceticacid
5 mglmI RNaseA (pancreaticRN;
RNase)
5x restriction bufferlRNase
III. Gel Electrophoresis
10><TrisIBoratelEDTA (TBE) electropboresisbuffer
Ix TrisIBoratelEDTA (TBE) electropboresisbuffer
0.8%, 1.0%, 1.5%, and 2% agarose
2.5% NuSieveagarose
Loading dye
5 mglml etbidium bromide stock solution
1 ~ml etbidium bromide staining solution
0.2% Methylene blue stock solution
0.025% Methylene blue staining solution
Iv. SouthemlColony
Hybridization
Denaturation buffer
Neutralization buffer
lOx sodium chloridelsodium citrate (SSC)buffer
2x SSCbuffer
2x SSCbuffel + 0.1% SDS
10% N-Iauroylsarcosine,sodium salt
Prehybridization buffer
Hybridization buffer
Washbuffer 1
Washbuffer 2
Washbuffer 3
Antibody/enzymeconjugatesolution
Color deveJopmentsolution
TrislIDTA (TE) buffer
-
v nNA I i~nnn
0.1 M adenosinetriphosphate (ATP)
lOx ligatiODbuffer + ATP
8
~
)
(
\
for Media,Rea&entS,
and~
Solutions
VI. BacteriaJ Transformation
1 M calciumchloride(CaQl)
so mM caliciumchloride(Ca~)
VII. Plasmid Minipreparation
0.5 M ethylenediaminetetraacetic
acid,disodiumsalt(EDTA)
GlucosetrrislEDTA(GTE)
S M potassiumacetate(KOk)
Potawumacetatelacetic
.cid
10% sodiumdodccylsulfate(SDS)
1% SDSIO.2
N NaOH..
VIII.
VIII. Polymerase
PolymeraseChain
ChainReaction
Reaction
0.9% sodiumchloride(N.Q)
SO mM Tris
1O% Cbe1ex
01l0roformf1SO8JDyl aIcobol
Pbenol (equilibrated)
PbenoVchloroformf1lO8JDyl aJcobol
3 M sodium acetate pH S.2
1 M potassium chlorlde (KQ)
1ax PCR buffu n
Deoxynuc1eotide lDixtuft
12.5mM and 2SmM ~
ABOUT
ABOUT
BUFFERS
BUFFERS
1. Solid reagenrsare typically dissolvedin a volwne of deionized or distiUed water equivalent to 70-80% of the finished volume of buffu.
This leavesroom for the addition of acielsor baseto adjust the pH.
FinaUy,water is addedto bring the solution up to the final volume.
2. When appropriate, the final concentration of each liquid reagent il
given in the right-hand column of the reagau list.
3. Buffen are used as2x, 5x, or 1ax solutions. Buffen are diluted what
mixed with other reagenrs,to produce a working concentration of
Ix.
4. The commercial enzymesusedfor theselaboratories are all supplied
with appropriate buffen; these should be used unless otherwiJe
noted.
S. Storagetemperaturesof ".C and -20.C refer to normal refrigerator
and freez.ertemperarura,
respectively.
tempe
3 89
390
Appendix2
m
""""""""
,...,
"
,..."..",..,
"
,
,.,
,
,..........
I. Bacterial Culture
4 N SodiumHydroxide(NaOH)
,
Makes 100 mI. Storeat room temperature(indefiniteIy).
1. Slowlyadd 16 g of NaOH pellets(m.w. 40.00) to 80 mi of deionized
nr
iIt1rrin,,"J1v.V\lnrinn urill
t--nnv. W'I'Y
warm-- tfiilt1l1M,.,atP!".
n
:r
n
---,
..---2. When the NaOH pellets are completely dissolved, add water to
make a final volume of 100 mi.
10 mg/mI Ampicillin
Makes100mi. Storeat -20.C for 1 rear or' at 4.C for 3 montbs.
1. 1\00 I g or amplClllm lSOOJWDsan, m.w. OR.j{ 1.1\11 ro IW
JJUor
deionizedor distilled water in a cleara2SO-mlflaak. (The sodium salt
dissolvesreadily; however,the free-acidform is difficult to dissolve.)
2. Stårto dissolve.
3. Prewasha 0.45- or O.22-micronsterile filter (Nalgeneor Coming) by
drawing tbrougb 5~100 mi of deionized or distilled water. Passthe
ampici1linsolution tbrough the filter.
4. Dispense100mIaliquots in sterile IS-mi tubes (Falcon 2059 or the
equivalent),and freezeat -20.C.
10 mg/mi Kanamyån
Makes 100 mi. Storeat -2o-C for 1 year or at -t.C for 3 montbs.
1. Add 1.0g of lcanamycin
sulfate(m.w.= 582.60)to 100 mI of deionizedor di.stilledwaterin a clean250-mIBask.
2. Stir to dissolve.
3. Prewash
a 0.45- or O.22-rnicronsterilefilter (1'Talgeneor Coming) by
drawing througb50-100 mI of deionized or distilled water. Passthe
kanamycin solution through the washedfilter.
4. DispenselO-mI aliquots in sterile lS-mI tubes (Falcon 2059 or the
equivalent),andfreezeat -20.C.
20 mg/mI X-Gal (S-Bromo-4-chloro-3-indolyl-p-o-galactoside)
Makes 100 mi. Storein the dark at -lo.e for 1 vear.
1. Add 0.5 g of X-gal (m.w. 408.63) to 25 mi of dimethylformamide
{m.w. 73.091in a cleanl00-ml fIaak.
2. Stil to dissolve.It is not necessaryto sterilizethe solution.
3. Dispense
l-mi aliquotsin 1.5-mItubes,and freczeat -200c.
LB broth can be consideredsterile as
1008 as the solution remainsc!ear;
doudiness is a sign of contamination by
microbes. AJwaysswirl the solution to
check for bacterial or fungal ceUstUt
may havesettledat the bortom of the
ftask or bottle.
Luria-Bertani (LB) Broth
Malcest liter. Storeat room temperature(indefiniteiy).
t. Weigh out:
t Og of tryptone
5 g of yeastextract
tO g ofNaQ (m.w. 58.44)
(Altematively, UR 25 g of pmnix, containing theseingredients.)
.
Recipes
for Med'1I,Reaeents.
and'StockSolutions
391
2. Add aUthe ingredientsto a dean 2-1Baskmat bas beenrinsed with
deionå.edor distilled wata:.
3. Add tl of deionå.edor distiUedwata' to the 6uk.
4. Add 0.5 mi of +M NaOH.
5. Stir to dissolve the dry ingredients, preferably uaing a magneåcstir
bu
6. If you are preparing for mid-Iog the cultwes, split the solution into
four 2S0-mI aliquotl in 1-1flaska.Plog the top with cotton or foam,
and cover it with aluminum foit. (Altematively, cover with cmIyaluminum foll.) Autoclavefor 20 minuaat Ut-c.
7. If you are preparing the solution for general usein transfonnatiom,
make 100-mI aliquOtl in sterile 1SO-2SO-mIbotdes, usingODeof the
following methodr.
a. Put on the caps loosely.Autoclave foc t5--20 minua at Ut.c.
(To help guard against breakage,autoclave the bottles in a sba1low pan with a small amount of wata:.)
b. Prewash
a 0.45-or O.22-niicronsterile filter (Nalgeneor Comin&)
by drawinsthrough 50-100 mi of deionized or disti1ledwater.
Passthe LB solution through the filter, and aliquot into sterile botdes.
Broth + Anuöioric
Antibiotic
LB~Broth
likestOO
mi. Store
Store at 4.C
3 months.
Makes
100mi.
4.C for 3,
months.
t. Sterilely add 1 mi of 10 mg/mi antibiotic to 100 mi of cooletl LB
,i
broth.
I
~. Add all mgred1ent5 to a clean ~-I nask that bU been nnse<l WIth
deionized or distilled wata:.
3. Add 11of deionizedor distilIedW81er.
4. AddO.smlof4NNaOH.
1,0 ",I
0,")1«) ~t
5. Stårto dissolve the dry ingredients, preferably using a magnetic Itir
bar. Anv undissolvedmaterial will dissolvedurina autoclaviD&.
6. Cover the mouth of the Baskwith alwninum fo~ and autoclave the
III\Intinn
fnr 1 ~ minntPC At 1'_18(".-
7. During autoclaving, the agar may settJeto the bortom of the fJasIc.
Swirl to mix the agar evenJy.
ytf =/-,
~
]92
,.
,
~2
"..................................................
8. Let the solution cool just until the flask can be held in your bare
hands (5~OC). (If the solution cools too long and the agar begins
to solidify, remelt it by briefly autoclaving for 5 minutesor less.)
9. While the agar is cooling, mark the bottorns of the culture plates
with tbc date and a description of the media (e.g., LB). If you are
using presterilized polystyrene plares, carefuUy cut the end of the
plastic sleeve,and saveit for storing the poured plates. Spreadthe
I
platesout on the lab bench.
.\
10. When the agar Baskis cool enoughto hold, lift tbe lid of the culture
plate just wide enoughto pour the solution. Do not set the lid down
on the lab bench.Quiddy pour in agar to just rover the plate bottom
(approximately 3 mm). Ttlt the plate to spreadthe agar,and immediately replacethe lid.
11. Continue pouring plates. Occasionally,flame tbc mouth of the flask
to maintain sterility.
12. To removebubblesfrom the surfaceof the poured agar,briefly touch
the surfacewith a burner flame while the agar is sti1Iliquid.
13. Allow the agar to solidify undisturbed.
14. If possible, incubate the plates lidside down for several houri or
ovemight at 37°C. This dries the agar, limiting rondensation when
the platesare stored under refrigeration; it also allows 3Oycontaminated platesto be readily detected.
15. Stackthe platesin their original plastic sleevesfor storage.
Antibiotic-containing platescan be
made quickJy by even1yspreading200
btI-of 100mglmlanribiotic on the surJ1l- face of an LB agar place.Allow the
anribiotic to absorb into the agar for
10-20 minutes before using. Ouwted
anribiotic platescan be refurbished in
this manna.
B Agar + Antibiotic
fakes 30-45 plates.Storeat 4°C for 2 months.
1. Follow the aboverecipefor LB agar plates, through step 9.
2. When the agar flau is cool enoughto hold, sterilely add 10 mi of 10mg/ml antibiotic. Ampicillin and kanamycin are destroyedby heat;
therefore, it is essentialto cool the agar before adding the antibiohc.
(For LBlamp+kanplates,add 10 mi eachof 10-mglml ampicillin and
kanamycin.)
3. Swirl the Baskto mix the antibiotic.
4. Resumewith step 10 above.
B Agar + Ampici1lin + X-Gal
:-gai is expensive.Therefore, we recommend spreadinga stock solution
f X-gal on the surfaceof premadeagar plates, rather man incorporating
le chemicaltbrougbout the enrlre plate. Store at 4°C for 2 montbs.
1. Prqme LB + ampicillin plates,as describedabove.
2. Add 40 J1I of the 20-mglml X-gal stock solution to a sterileLB plate +
ampicillin.
3. Use a sterile spreading roo to evenly distribute the X-gal over the
enrlre surfaceof the plate.
4. Incubate the plate(s) at 3~C until all the liquid has evaporated
(about 3 or" hours).
.
~ces
for Media.ReaPents.
andStockSolutions
StabCultures
Makes J()
40
stabs.Storein the dark at room temperaturefor t rear.
t. Weighout:
1.0g of tryptoae
0.5 g of yeut
extr8Ct
t.O g of Naa (m.w.58."")
t.Ogofapr
2. Add all ingredientsto a dean 250-mI Rask that bas beenrinsed with
deioniud or distilled water.
J. Add tOOmi of deionizedor disti11edwater.
4. Add SOJ11of -iN N.OH
S.Stirwhilehearingto dissolvethe dry ingredients, prderably using.
magnetichot plateandstir bE
6. Pourthe dissolvedsolutioninta 4-mI vials (15 x 45 mm)to fin the
vial two-thirdsfull, andlooseIyreplacethecape.
7. Autoclave the vials for 15 mimJtesat 1218C.{Alternativety, sterilir.ed
agar can be poured inta presterilizedvials.)
8.Allow the agar to solidify undisturbcd.
9. Bdorestoring the solution, tighten the capssecureIy.
10. SeaIthe caps with ParafiIm to help reduce the danserof contaminatioo.
To inocuJa~:
1. Stcri1elyscrape up . ceUmalS hom 8 single oolony of the desired
genotype.
2. Staban inocularing loop severaltimea into the agar.
3. LooseIyreplacethe cap, and incubate the stab overnight at 3'-C.
4. FoUowingincubation, tighten the cap, and store in the dark at room
temperature.Wrap the cap with parafilm for long-term sto....
II. DNA Restriction
1 M Tris (pH 7.6, pH 8.0, and pH 8.3)
Makes 100 mI- Storeat room mnneratDl'l!
tinddinitdvt
1. Dissolve 12.11 of Tris base(m.w. 121.10) in 70 mi of deionized or
distiUedwater.
2. Adjust the pH by slowly adding concentratedhydrochloricaåd
(Ha); monitorwith a pH mecu.
3. Add deionizedor distilledwater to makea total of 100 mi of solutioa.
Avold InhallnaTris powder;war a maskover ~ur noseand mouch.
] 9]
This recipeis the sameas for the LB
agar above, bur it hasa lower pem:ntagc of .r, which nlakes the Stabe3sier
to UK. StandardLB agar or 4 g of ptTmix can 31$0be osed.
194
Appendix2
Nates:
a.A yellow solution indicates poor-quality Tris. Discard the solution,
and obtain Tris from another source.
b. Many types of electrodesdo not accurately measurethe pH of Tris
solutions; check with the manufacturer to determine whether your
electrodeis Tris-compatible.
c. The pH of a Tris solution is temperature-dependent;make pH measurementl at room temperature.
5 M Sodium Chloride (NaCl)
Makes 100 mi. Storeat room temperature(inddinitely).
1. Dissolve 29.2 g of NaCl (m.w. 58.44) in 70 mi of deionizedor distilled water.
2. Add deionizedor distil1edwafer to make 100 mi of total solution.
1 M MagnesiumChIoride (MgCll)
Makes 100 mi. Storeat room temperature(indefinitely).
1. Dissolve 20.3 g of MgCll (6-hydrate, m.w. 203.30) in 80 mi of
deionizedor distiUedwater.
2. Add deionizedor distil1edwafer to make100mi of total solution.
1 M Dithiothreitol (DIT)
Makes 10 mi. Storeat -20.C (indefiflitely).
Do not autoclaveDIT or solutions
containing it.
1. Dissolve I.S g of DIT (m.w. 154.25) in 8 mi of deionizedor distilled
wafer.
2. Add deionizedor distil1edwafer to make 10 mi of total solution.
3. Dispense
inta t-mI aliquotsin l.S-mI'rubes.
10><CompromiseRestriction Buffer
Makes 1 mi. Storeat -20.C (indefiniteiy).
100J1l of 1 M TrispH 8.0
l00J1loflM~
200 J1lof 5 M NaO
7 J1lof 14.3 M pME
(100mM)
(100mM)
(1 M)
(100mM)
593 J1lof deionizedwater
or
tOOJ1I of t M Tris pH 8.0
tOOJ1I of t M~
200 J1I of S M Naa
tO J1I of t M DIT
S9OJ1I of deionizedwater
Mix the ingredientsin
in a
a 1.5-ml
t.
robe.
(100mM)
(100mM)
(1 M)
(10mM)
8
~
for Media.R.eaIents.
andStockSolutions
Nota:
a. ~mercaptoethanol (PME), aJsocaUed2-mercaptoethanol,il. 14.3M liquid at room temperatuR.
b. Compromise buffel provides salt conditions that aDow relatively
high activity of a number of restriction enzymes,inc1udingmost of
those used in theselaboratories. Consider usins tbc buffer providcd
by the manufacturer, wben you are using mott expensiveenzymesin
critical experimeDtI.
c. DIT used in the seeondrecipecan precipitate from the solution with
repeated
freezing and thawiJJa.Vortex vigorously to redissolvethe.
DTI'.
lOx NEBuffer 1 (yeDow)
Makes 1 mi. Store at -2o-C (indefiniteIy).
100 JI.Il M Tris propane pH 7.0
100 J&11M
10 J&11M DIT
790 J&1
distilled water
~
(100 mM)
(100mM)
(10 mM)
lOx NEBuffer .. (green)
Makes 1 mi. Store at -2o-C (ind~nitely).
200 J&11M Tris-acetatepH 7.9
100 JI.Il M magnesiumaceta1le
500 J&11M potassium aceta1le
10 J&11M DIT
190 J&1
distilled water
(200 mM)
(100 mM)
(500 mM)
(10 mM)
30 mM S-AdenosylMethionine (SAM)
Makes 1 mi. Store at -2o-C fcc 1 JMr.
1. Obtain 1 M suJfuric acid (HaSO.), or prepare by carefu1lyadding 1
part concentrated acid (18'M) to 17 parti of deionized or distilled
W8Ia'.
2. Preparea S-mM solution by adding S J&1
of 1 M HzSO. to 99S ~ of
deioniz.edor distilled W8Ia'.
3. Add900J&1 of 8 S-mM HaSO.solution to 100 J&1 of 100% ethanoL.
4. Dissolve 15.8 mg of SAM (iodide salt, grade I, m.w.
= 526.3)
in 1 mi
of a S-mM HzSO/IO% ethanol solution.
5. Dispense tOO-~ aliquors in t.S-mI tubes. Use esch solution once,
and then discard it.
Nota:
8. SAM il very unstable, and ita activity diminishesrapidly with freezing and thawing. Store SAM solution on ice at all times; thesesolutions have a half-life of about 30 minutes at room temperature.
b. 30 mM of SAM is oftensuppliedwith M.Ec:oRImethylue.
395
396
Appendix
2
"
'
2x Rcstriction BuffL'f
Makes 1 mi. Storeat -20.C (indefiniteiy).
Mix a 200-J1l1Ox restriction buffer with
with 800
800J1l
JU of
of deio!
deionizedor distilled
water.
~C;rt lI"'-1
,
0.0$%
0.0$% Glacial
Glacial Acetic
Acetic Acid
Acid
so.J.
., t>pc.,°/. 'J \,L-1~.'1
Makes 50 mi. Storeat room temperature(indefiniteiy).
1. Add 5 J1l of glacial aceticacid to 50 mi of deionized or distillcd water.
2. ThesolutionshouldbeapproximatelypH 4.0.
I
5 mg/mi RNaseA (PancreaticRNase)
Makes 20 mi. Storeat -20"'C (jrWfinitely).
1. Dissolve100 mg of RNaseA in 20 mi of 0.05% glacialaceticacid,
and transfer the solution to a 50-mI conical robe.
2. Placethe robe in a boilinsrwater barnfor 15 minutes.
.
3. Cool the solution, and neutralizeit by adding 120 ~ of 1M Tria (pH
8.0).
4. Dispensel-mi aliquotsin l.S-mI tubes.
Notes:
8. UIe only RNaseA &om bovine pancreas.
D. UISSOIVIDg 1U\Ia5e m acetIC aCIQ preveDts suosequent
preapttatlOD
01
the RNase.The solution can be preparedby simply dissolvingRNase
in deionizedor disti.lledwater; howeveJ;the RNase will ocx:asionally
precipitate from solution, and activity will be lost.
c. The boiling step destroysthe activity of DNases that can contaminate the preparation. The RNaseactivity will not be affectedby boilmg.
(
Sx Restriction BufferlRNase
Makes1 ml. Storeat -lO.e for severalmontbs.
.,es~1h1Yn:"~'(
:f
. i fll( t
,.oJ.({
500 ~ of tO><restriction buffer
tOO~ of S-mglmI RNase
400
J...o\<
tf t ! f' i.,,1IIi
~ of water
Mix in a t.S-mI tube.
III. Gel Electrophoresis
lOx Tris/Borate/EDTA (TBE) ElcctrophoresisBuffer
~he1 ~ ~Wlt~
Makes 1 liter. Storeat room temperature(indefiniteiy).
1 g of NaOH (m.w. 40.00)
108 g of Tris base(m.w. 121.10)
SSg of boric acid (m.w. 61.83)
7.4 g of ethylene diamine tetraacetic acid (EDTA, disodium salt, m.w.
372.24)
,
C .
-
Recipes
for Media,~
(:
andStockSolutions
1. AddaUdry ingredientsto 700mi of deioniz.ed
or distilledwaterin a
2-lflask.
2. Sricto dissolve,preferably using a magneticsric bar.
3. Add deionizcd water to bring the total solution to tlitu.
1x TBE Elearophoresis Buffcr
Makes 10 liten. Store at room temperature(inddinitdy).
t. Into a spigotedcarboy,add9 liten of deionizedor distilledwaterto
t literof lO><
TREelectrophoresis
buffu.
2. Stir to mix.
0.8%, 1.0%, 1.5%, and 2.0% Aprose
Makes 200 mi. Use &es~ or store jeUedat room temperature for severa1
weeb.
1. Add 1.6 g (0.8%),2.0 g (1.0%), 3.0 g (1.S%),or 4.0 g (2.0%) of
agarose(dectrophoresis
grade)to 200 mi of 1x TBEdectrophorais
bufferin a 600-mIbeakeror ErIeomeyer
fIuk.
2. Sar to suspendthe agaroee.
3. Cover the container with aluminum foit, and heat in a boiling water
bath (use a double boiler) or on a hot pla~ until all the agaroeeis
dissolved(approximatdy 10 minuta).
or
Heat uncavered in a microwave aven at the high setting, untiJ all the
agaroseis dissolved(3-5 minuta percontainer).
4. Swirl the solution, and check the honom of the container to enaW'e
that aU the agarosehas dissolved.Uust heforecompleredissolution,
particles of agarosewiU appeal as translucent grains.) Reheat for
several minutes. if necaI8I'Y.
S. Coverthe solution with aluminumfoil, and placeit in a hot wafer
bath(at about6Q-C)until you arereadyto WIeåt.
Nota:
a. Samplesof agarosepowder can be preweighed and stared in capped
test tubes until you are ready to usetbem.
b. If you are microwaving agarosein a beakeJ;a .skin. of solidifed
agarosemay form on the surface.Removethis skin before pourinl a
Fl
c. Solidified agarose can be stored at room temperature and then
rerneltedin a boiling water barn (15-20 minutes) or in a microwave
oven (3-5 minutes per container) before UIe. AJways l00sen the cap
wben remdting agarosein a bottle.
d. When remelting agarose,evaporation will causethe concentration to
increase. Il necessary,compensate by adding a small volume of
watu.
397
398
Appendbc2
2.5% NuSieveAgarose
Makes 200 mi. Use fres~ or store jelled at room temperaturefor several
days.
1. Add S g of NuSieveagaroseto a 200-mllx TBE electrophoresis
buffer.
2. Stir to suspend
theagarose.
- .
. -.
. -"3. Cover the container WIth aJummumtOlI, and heat m a bOtJJng
water
bath (use a double boiler) or on a hot platet until all the agaroseis
dissolved(approximately 20 minutes).
or
Heat uneoveredin a microwaveaven at the high setting, until all the
agaroseis diaolved (3-5 minutesper container).
4. Swirl the solution, and check the bottom of the container to ensure
mat aU the agarosehas dissolved.GUlt before complete dissolution,
partides of agarosewill appear as translucent grains.) Reheatfor
.
several minutes, if necessary.
5. Cover the solution with aluminum foit, and place it in a hot water
bath (at about 60.Q until you are ready to use åt. Remove any
lOskin" of solidified agarosefrom the surfacebefore pouring.
Loading Dye
Makes 100 mi. Storeat room temperatute(indefiniteiy).
0.25 g of bromophenol blue (m.w. 669.96)
(0.25 g of xylenecyanol(m.w.538.60» RY.""", 14.,
50.00g of aucroee(m.w.~2.30) or 50 mi of glycerol
1.00 mi of 1 M Tris (pH 8.0)
If vou are usina sucrose:
1. Dissolve bromophenol blue, xylene cyanol, sucrose,and Tris in 60
...1 ,..f A.-;,
; ,J ,
A;
II..,J n...-
2. Add deionizedor distilled water to make 100 mi of total solution.
Tf vnn ar,. l1!ilinsr
--Illvcf'rnl:
.
1. Oissolve xylene cyanol, bromophenol blue, and Tris in 49 mi of
-'.'--'---'
~V~
-- -',_--,II_-'_._~--
VA
u
"'."-&0
2. Stir in 50 mi of Illvcerol to make 100 mi of total solution.
5 mglml Etbidium Bromide Stock Solution
Makes50 mI. Storein the dark at room temperatureor at 4°C (indefiniteIy).
e
1
Recipesfor Mecfra,
Reagents,
andStockSolutions
399
1
l
'j
~
Ethidium bromide is a mutagenby the Ames microsome issay and a suspect8d
carcinogen. Becausemlxing the 5 mg/mi solution from the concentrated powder poses the greatest hazard.we recommend obtalnlng ready-mlxed 5 m&lml
solution from a suppller. If you choose to mix ethldlum bromide solution. hMdie the powder carefully to avold creating dust. War rubber gtovesanda mak
that covers your nose and mouth. Observe the proper disposaImethocå
explalned on pace 38.
1. Dissolve 250 mg of ethidium bromide in 50 mI of deionized or dis.
tilled water in a nonbreakable, screw-top container (preferably
opaque).
2. Label the bottle "CAunON: Ethidium Bromide. Mutagen and can-
cersuspect
agent.
Wearrubbergloves
whenhandling.
.
Ethidium bromide is light-sensirive;
store it in a dark container, or elsewrap
the container in aluminum foiJ.
1 ).1l/mIEthidium Bromide StainingSolution
Makes 500 mi. Store in the dark at room temperature (indefinitåy)
Ethidlum bromide Is a mUtagenby the Arnes microsome assayand a suspecr8d
carcinogen. Wear rubber gtoveswhen preparing and uslng ethidlum bromlcle
solution. Observe the proper dlsposaJmethods explalned on page 38.
1. Add 100J1l of a 5 mg/mIethidium bromide solutionto 500 mi of
deionizedor disti1ledwater.
2. Storein unbreakablebottles(preferablyopaque).Labelthe botta
"CAUI10N: EthidiumBromide.Mutagenandcancersuspectagent.
Wearrubbelgloves
whenhandling.
.
0.2 % Methylene Blue Stock Solution
Makes 100 mi. Store at room temperature(indefinite1y).
1. Weigh out 0.2 g of methyleneblue-trihydrate (m.w. 373.9), and add
it to 100 mI of distilled water.
2. Sår until it is completelydissolwd.
0.025% MethelyeneBlue StainingSolution
Makes 500 mI. Store at room temperature(inddinitely).
1. Add 62.5 mI of 0.2% methelyene
bluestocksolutionto 437.5mI of
distilled water.
2. Stil to mix the solution.
Ethidium bromide is light-sensitive;
store it in a dark container, or dse wrap
the container in aluminum foil.
~
Rcview the commentSrcgarding Tris
solutions on pa~cs393-394.
8
60.6g of Tris (m.w.121.10)
(0.5M)
87.7g of NaCI (m.w.58.44)
(1.5M)
2. Stirto dissolve,preferablyusinga magneticstir bar.
3. Adjust the pH to 7.5 by slowly addingconcentratedHCI; monitor
with a pH meter.
4. Add waterto bringthetotal solutionto 1 liter (1,000mI).
JOxSSCBuffer
Makes tlirer. Storeat room temperature(indefiniteiy).
1. Add the following dry ingredientsto 700 mI of deionizedor distilled
water:
87.7 g NaO (m.w. 58.44)
(1.5 M)
44.1g sodiumåtrate (m.w.294.1)
I
(0.15 M)
2. Stir to dissolve,preferably using a magneticstir bar.
3. Adjust pH to approximately 7.0 by adding NaOH solution.
4. Add watetto bringthetotal solutionto 1 litet (1,000mi).
2x SSCBuffcr
Makes 1 liter. Store at room temperature(indefiniteiy).
1. Add 200 mi of 10x SSCbuffer to 800 mi of deionized or distilled
water.
2. Stir to mix.
2x SSCBuffer + 0.1% SDS
A prccipitatc may form at calder temperatUrl'S.Warm the solution, and
shah' it gently tO dissolveth~'pTl'\:ipi.
tate.
Makes 1 liter. Storeat room temperature(indefinirely).
1. Mix 10 mI of 10% SDSwith 990 mI of 2x SSCbuffer.
2. Stir to mix.
Recipesfor Media,Reagents,
andStockSolutions
1
40 I
"",""""""""""""""""""""""""""""""""
10% N-Iauroylsarcosine,Sodium Salt
Makes 100 mi. Storeat room temperature.
~
~
.
1. Dissolve 10 g of N-lauroylsarcosine, sodium salt (m.w. 293.4) in 80
mi of deionizedor distilled wa~
2. Add water to make 100 mi of total solution.
Prehybridization BuHer
Makes 100 mi. Store at -20.C for 1 yeu.
1. Mix the following in a 200-mI fJuIc:
50 mI of lOx SSCbuffa1 mI of 10% N-lauroylsarcosine
(5x SSC)
(0.1% wIv)
200 JU of 10% SDS
(0.02%wIv)
30 mI of distilled wata'
2. Add 1 g of blocking reagent (supplied with the Genius Detection
Kåt).
3. Dissolvethe blocking reagentby stirring at So-70.c, preferably
usinga magneticsm bar.Thesolutionwill becometranslucent.
4. Add waterto make100mi of total solution.
,
Preparethe solution in advance,and
cool it to below 40.C befoR UJe.
Hybridization Buffer
'f' i
Makes 100 mi. Store at -20.C for 6 months.
Add a I@kd nlignnnrIPl\,.;tf..
prnh..to 100 mi of prehybridizationbu.ffer,
so that the probe concentration is about 10 nglml.
WashBuffc:r1
..
. 4lJ).."
l~
2"1)jJ .6li
~.~~{
..:
f
(
-::'1!'~'~f)
Makes 1 liter. Store at room temperature (indefinitely).
1. Mix the following in a 2-1fIuk:
100 mi of 1 M Tris pH 7.6
30 mi of S M Naa
870 mi of deionizedor distilled water
(100 mM)
(150 mM)
,
2. Stir to mix.
Wash Buffer 2
Makes 1 liter. Store at -2o-C for 1 year.
1. Add 10 g of hlocking reagentlsupplied with the Genius Detection
Kit) to 1,000 mI of wash buffer 1 in a 2-1flask.
2. DissoJvethe blocking reagent by stirring it at S0-70°C, preferably
usinga magneticstir bar. The solution will becometranslucent.
~
WashBuffer3
,
Makes t liter.Storeat room temperature(indefinite1y).
-
-
. 1. Add 12.1 g of Tris (m.w. 121.1) to a 2-1 flask containing 700 mi of
deionizedor distilled water.
2. Stir to dissolve,preferably using a magnetic sår bar.
Prepare the solution in advance,and
cool it to below 40.C before use.
402
402
"...,',
,.,""""""',.,"'~.,~"..,",...,
,"","""""""""""""""""""""""""""""""""""""'",
".............
3. Add 20 mi of S M NaCl.
4. Add SOmi of 1 M MgC~.
S. Adjust the pH by slowly adding concentrated HCl; monitor with a
pH meter.
6. Add water to make 1000 mi of total solution.
Antibody/Enzyme Conjugate
Makes 100 mi. Storeat 4°C for oo1y12 houn.
Add 10 JLl of the ~hodv/enzvme con!::fate (supplied with the Genius
Detection Kit) to a Baskcontaining 100 of wash buffer 2, and mix.
Color DevelopmeotSolution
Makes 10 mi. Preparefresh solution.
Once the color dcvdoprnmt solution
has beenmixed, åtshould be stored in
the dark.
To a flask containing 10 mi of wash buffer 3, add 4S JUof nitroblUl! ~
zolium
salt
and 3S
JU of X-Dh~phate solution; both of theseare supplied
With
the
Genius
Detection
Kit.
-
@
TrislEDTA (TE) BuHer
~~
(;/t l'l4,~
l~'iir~
c"~I~)
Makes 100 mi. Storeat room temperature(inddiniteJy.)
1 mi of 1 M Tris (pH 8.0)
200 JUofO.SMEDTA
99 mi of deionizedwater
(10 mM)
(1 mM)
Mix the ingredients.
V. DNA Ugadon
0.1 M AdenosineTripbosphate(ATP)
8
Makes 5 mI. Storeat -lOoe for 1 rear.
1. Dissolve 0.3 g of ATP (disodiwn salt, m.w. 605.19) in 5 mi of deionized or distilled water.
2. Dispense
SOO-JlIaliquotsinto 1.S-mIrobes.
lOx Ligation Buffer + ATP
Makes 1,000 J.1l. Storeat -20.C for 1 month.
600 J.1l of 1 M Tris, pH 7.6
100 J.1l of 1 M ~
70 J.1l of 1 M DIT
100 J.1l of 0.1 M ATP
(600 mM)
(100 mM)
(70 mM)
(100 mM)
130 uj of deionizedwater
Mix theinwedientsin a l.S-ml tube.
Notes:
a. DIT can precipitate from the solution with repeated freezing and
thawiJ1s.Vortex vigorously to redissolve.
b. ATP losesactivity in a dilute solution; be sure to usefresh solution.
Redpes
for Media.Reagents.
andStockSolutions
403
-'-H""""""""""""""""""""""""""""H"""""""'"""""""""""""""""""""""""""""""""""""""""""".....................................................................
VI. Bacterial Transformation
1M Calcium Chioride (CaC2)
Makes 100 mI. Store at room temperature(indefinitdy).
1. Dissolve 11.1 g of anhydrous CaQl (m.w. 110.99) or 14.7 g of the
dihydrate (m.w. 146.99) in 80 mi deionizedor distilled water.
2. Add deionized or distiUedwater to make 100 mi of total solution.
50 mM Calcium O1loride (CaCl2)
Makes 500 mI. Store at ".C or at room temperature(indefiniteiy).
1. Mix 50 mI of 1M Ca~ with 950 mi of deionizedwater.
2. Prerinse a 0045- or 0.22-micron sterile filter by drawing through it
50-100 mi of deionizedor distilled water.
3.Passthe Ca~ solutionthrougha prerinsedfilter. .
4. Dispense
aliquots
into
presterilized
SO-mi conical
robes or auto-
claved 1S0-lS0-mI bo..
Alternatively.
dispeme100-mlaliquOCll
into 150-:-250-mlbotdes. and autoclave
for 15 nunutcs ar 1218C. With 5torage
ar 48C, the solution will be precooled
andreadyfor makinftcomJX-rmt
cells.
VII. Plasmid Minipreparation
0.5 M Ethylene Diamjne TetraaceticAåd (EDTA) (pH 8.0)
Makes 100 mI. Store at room temperature(inddinitely).
1. Add 18.6 g of EDTA (disodium salt, m.w. 372.24) to 80 mi of deionized or distilled wata'.
2. Adjust the solution to pH 8.0 by slowly addingsodiumhydroxide.
Monitor with a pH meta".
3. Mix vigorously using a magnetic stirrer or by hand. EDTA will dis-
solveonlyaherthepH hasreached
8.0or highcr.
GlucosetrrislEDTA (GTE)
(AIiP~
~
~
UIeo.,
" Ji!«!iWII :hIttof t::OTA.
f,A~<1
~rf
p""J~
Makes 100 mi. Store at -J.C or at room temperature(indefinite)Y)'rl1Ii'i»~
0.9g of glucose(m.w.180.16)
2.5 mi of 1 M T~ (pH 8.0)
2 mI of 0.5 MEDTA
(SOmM)
(25 mM)
(10 mM)
94.5mi of deionizedwala'
~~
.
(~
,el.
tV./IJtr.
Whcl1smred at 4-C, ..h.:solution will
~1f' fk,61!) be pr~II()1edand I'eöldyfor miniprcparatIon!'.
S M PotassiumAcetate (KOAc)
Makes 200 mI. Store at room temperatuR (inde6nitely).
1. Add 98.1 g of potassiumacetate(m.w. 98.14) to 160 mI of deionized
waur.
2. Add deionizedor distilled water to make 200 mI of total solution.
PotassiumAcetatelAcetic Acid
Makes 100 mi. Store at 4.C or at room temperature(indeMirely).
Add 60 mi of S M potassium acetate and 11.5 mI of glacial acetic acid to
28.5 mi of deionized or distilled water.
The sharp edor of acericarid distinguishesthc 6nishedKOAclacetic arid
solution from the KOAc srock; the tWo
can be easily confuSt.-d.
When stored at
.. -C, the solution will be precooled and
re:1dyfor miniprep3l'3tionS.
~
10% Sodium Dodecyl Sulfate (SDS)
Makes 100 mi. Storeat room temperature(indefiniteiy).
~I,! .
'J";), .
"/""
f"!(' ..~l.
,
l
1. Dissolve 10 g of electrophoresis-gradeSDS(m.w. 288.37) in 80 mi of
deionizedwater.
2. Add deionizedor distilled water to make 100 mi of total solution.
1,';\.
",ilc
~Ir'.:.!
sosIs the sameas sodlum lauryl sulfate. Avoid InhallngSOSpowder: wear a
mask over your nose and mouth.
1% SDSlO.2N NaOH
Always uscfresh SDSlNaOH solution.
A prccipitate can fonn at calder temperatures.Wann the solution and
shakeit gently,to dissolvethe precipitate. 11
. _..~I<
) r'r'
'
I '-
'~'-"'
"' "
IN)
.(~! I
.
.
f
.1I') .~~ (
i
'.'
1"<
.
j
(tJj
"Ii 1-1/(;
r
Makes 10 mi. Storeat room temperaturefor severaldars.
8
Mix 1 mi of 10% SDSand 0.5 mi of 4 N NaOH into 8.5 mi of distilled
water.
.'k,;ltUt.r~ <. ~(,..~
VIII. Polymerase Chain Reaction
0.9% NaCI
> 11'.'.,
'. . I c .'
"
If the solution is to be usedimmediately, it doesnot needto be sterile.
However, for long-tenn storage,we
recommendsterilizing by autodaving it
for 15 minutes at 121°C or passingit
through a 0.45- or O.22-micronsterile
filter.
Makes 100 mi. Storeat room temperature(indefinitely, if sterile).
1. Weigh out 0.9 g of NaO (m.w. 58.44).
2. Add it to a dean 200-mI flask containing 90 mi of deionizedor distilled water.
3. Stir to dissolvethe NaCI, preferably using a magneticstir bar.
4. Add water to bring the total solution to 100 mi.
50 mM Tris
Makes 100 mI. Storeat room temperature(inddinitely).
Dissolve 0.6 g of Tris base (m.w. 121.10) in 100 mi of deionized or distilled water. The solution will havea pH of about 10.
10% Chelex
Makes 10 mI. Storeat room temperaturefor 3 months.
1. Weighout 1 g of Chelex100 (100-200 mesh,sodiumform, from
BioRad).
2. Add SOmM Tristo thedry Chelex,to make10 mi of solution.
3. AdjustthepH to 11,usingconcentrated
NaOH.
Chloroform/Isoamyl Alcohol
Makes 100 mi. Storeat room temperature(indefiniteiy).
lsoamyl alcohol is also called isopentyI
alcohol.
Add 4 mI of isoamylalcohol(m.w.88.15)to a dean bottle containing96
mI ofchloroform (m.w.119.38),andmix.
IJ!>.
\'!W
. ~
.'
. !
1
Redpes
for Media.Reagents,
andStockSolutions
405
..........................................................................................................................................................................-....................................................................
)
.
Phenol(Equilibrated)
Makes 100 mI. Storeat 4.C for 1 month.
.
l
1. Remove the liquified phenol (m.w. 94.11) &om the freezer,warm it
to room tempera~ and then mdt it at 6S8c.
2. Add 50 mI of themeltedphenol to a light-tightbotde.
3. Add 50 mI of 0.5 M Tris pH 8.0, and stir for IS mmutes, preferably
using a magnetic stir bu:.
4. Allowthe
two phases to separate, and aspirate as much of the upper
aqueous phase as possiblc.
5. Add SO mi of 0.1 M Tris pH 8.0 to the phenol, and stir for 15 minDta.
6. Removethe upper aqueousphue, as describedin step -t.
7. Repeatextractions with SOmi 0.1 M Tris pH 8.0 until the pH of the
phenolic phaseis greaterthan 7.8 (asmeasuredwith pH paper).
8. Add a final SOmi of 0.1 M Tris pH 8.0 to the pheno1.
Phenol Is corrosive and can causesevere bums. Be sure te handle phenol
",nder a chemica/ hoad; war protectlve c/ottling, gIoves. and safety gJasses.
Rinse with a large volume of water any area of skin that comes Inte contact
with phenol. Wash with scap and water. Do not use ethanol.
,
I
,
PhenoVChloroformlIsoamyl Aleohol
Makes 100 mi. Store at 4.C for 1 montb.
Add SOmi of equilibratedphenolto a dean, light-tight bottle containing
SOmi of ch1oroformflSoamyl
aleohol,andmix.
3 M Sodium Acetate
Makes 100 mi. Store at room temperarure(indefinitely).
1. Dissolve 40.8 g of sodium acetate(m.w. 136.08) in 70 mi of deion-
izedor distilledwacu
2. Adjust the pH to 5.2 by addingglacialaceticarid; monitor with a
pH meta".
3. Add water to bring the total solution to 100 mi.
1 M PotassiumCh1oride(KO)
Makes 100 mi. Store at room temperature(indefiniteiy).
1. Dissolve7.46 g of potassiwncblondein 70 mi of deionizedor distilled water.
2. Add waterto bringthetotal solutionto 100mi.
We do not recommendusing crystalline
phenol. beca~ it must be rectistilledto
removeoxid~1tionprodut.'tsthat can
damageDNA.
406
",...,
"
~
Appendix2
"""""""""""""""""""""""""""""""""'".........................................................
10><PCR Buffer II
Makes 1 mi. Storeat -loDe (indefiniteiy).
100J&1 of 1 M TrispH 8.3
500 J&1 of 1 M KO
400 J&1 of deionizedwater
(100
(100m
mM)
(500
m
(500mM)
Mix the ingredientsin a lo5-mI tube.
Deoxynudeotide Mixture
Makes 1 mi. Storeat -loDe for 1 rear.
10mM dATP
10mM dCTP
10mM dGTP
125
125J&1
~ (1.25mM)
125
125J&1
~ (1.25mM)
125
125J&1
~ (1.25mM)
10mMdTIP
125J&1
125~ (1.25mM)
deionized~ O
500
J&1
500~
Mix in a 1.S-mItube.
12.5 mM and 25 mM MgC12
Makes 1 mi. Storeat -loDe for 1 rear.
Mix ll.5
J&1
of 1M MgC(z with 988 J&1
of deionizedwater.
(12.5mM)
or
Mix 25 J&1
of 1M
~
with 975J&1 of deionizedwater.
(25 mM)
8.