METHODS HANDBOOK
Department of Molecular Biology
Massachusetts General Hospital
Department of Molecular Biology
Methods Handbook
Table of Contents
A. Glasswashing and Sterilization
I. General Glasswashing
4
II. Pipette Washing
4
B. Electrophoresis Buffers
I. Tris-Acetate (TAE)
5
II. Tris-Borate (TBE) [normal strength]
5
III. Tris-Borate (TBE) [low strength]
6
C. Liquid Media
I. LB (Luria-Bertani) Medium
7
II. M9 Salts for Medium
8
III. TY Medium
9
IV. YPD Medium
10
D. Plates
I. LB (Luria-Bertani) Plates
11
II. TY Plates
12
III. YPD Plates
13
IV. RNAi Plates
14
V. Standard Worm Plates
15
VI. Enriched Worm Plates
16
E. Antibiotic Plates
I. Ampicillin (Amp). Includes AMP/TET.
17
II. Chloramphenicol (Cam)
18
III. Kanamycin (Kan)
19
IV. Neomycin (Nm)
20
2
Department of Molecular Biology
Methods Handbook
V. Streptomycin (Sm)
21
VI. Tetracycline (Tet)
22
F. Stock Solutions
I. 20 % Ammonium Sulfate (20 % (NH4)2SO4 )
23
II. 10x BS
23
III. 1M Calcium Chloride (1M CaCl2)
24
IV. 20 % CAS Amino Acids
24
V. 40 % Glucose
25
VI. 10x Nif-P
25
VII. 10x Phosphate (PO4)
26
VIII. 40% Sucrose
26
IX. 5M Sodium Chloride (5M NaCl)
27
X. 2N Sodium Hydroxide (2N NaOH)
27
XI. Sodium Molybdate Stock (Na2 MoO4 )
27
XII. Water (Sterile)
28
XIII. 1 M Magnesium Sulfate.
28
XIV. I M Calcium Chloride
28
XV. 1 M Potassium Phosphate
29
XVI. 5 mg/ml Cholesterol
29
XVII. S Basal Solution
29
XVIII. Streptomycin
30
XIX. Nystatin
30
XX. M9 Buffer
30
XXI. OP50 for worm plates.
31
3
Department of Molecular Biology
Methods Handbook
A. Glass washing and Sterilization
1. General Glassware
Dirty glassware is placed in the Better Built Turbomatic washer and washed on
the normal cycle (30 minute run, 10 minute wash) with an extra 10 minute
extended (third) rinse cycle. No glassware is washed without the extended
rinse. Beakers and cylinders are removed at this point. These items may be dried
further in the high temperature oven upon request by specific lab groups, as are
certain large flasks.
Remaining glassware is capped with aluminum foil and sterilized in one of the
large AMSCO autoclaves. Sterilization is at 250° F for 20 minutes on gravity
cycle with an additional 15 minutes on dry cycle.
A. Glass washing and Sterilization
2. Glass pipettes
Pipettes are soaked initially in hot water for a few minutes, then flushed with
warm tap water for a minimum of 15 minutes. After allowing the water to drip out for
several minutes, the pipettes are soaked in cleaning solution Micro for a minimum of 30
minutes (usually 1 hour). After allowing the cleaning solution to drip out for several
minutes, the pipettes are flushed in tap water for a minimum of 30 minutes. Next, they are
flushed in pure water for 15 minutes, separated by size, and placed into stainless steel
holders. The pipettes are dried in these holders for a minimum of 4 hours (usually 10
hours overnight) at 220° C.
4
Department of Molecular Biology
Methods Handbook
B. Electrophoresis Buffers
1. Tris-Acetate (TAE) Buffer-50x Stock
Into a Nalgene plastic or glass volumetric beaker, add approximately 3/4 of the
final required volume of pure water. Add the following items in the amounts
corresponding to the final solution volume you wish to make from the table
below: Trizma Base (reagent grade), Glacial Acetic Acid, EDTA-disodium salt.
Stir with the help of a stirring bar until dissolved (ca. 20 min) and dilute to the
desired volume with pure water. Stir again and check pH with 7.5-14 pH paper.
Correct reading should be 8.5-9. Transfer to 1 L Wheaton bottles.
Reagent
1 Liter
trizma (tris) base
242 grams
glacial acetic acid
57.1 mLs
EDTA-Disodium salt 37.2 grams
2 Liter
3 Liter
4 Liter
484 grams
114.2 mLs
74.4 grams
726 grams
171.3 mLs
111.6 grams
968 grams
228.4 mLs
148.8 grams
B. Electrophoresis Buffers
2. Tris-Borate (TBE) Buffer - NORMAL 10x Stock
Into a Nalgene plastic or glass volumetric beaker, add approximately 3/4 of the
final required volume of pure water. Add the following items in the amounts
corresponding to the final solution volume you wish to make from the table
below: Trizma Base (reagent grade), Boric Acid, EDTA-disodium salt. Stir with
the help of a stirring bar until dissolved (ca. 20 min) and dilute to the desired
volume with pure water. Stir again and check pH with 7.5-14 pH paper. Correct
reading should be ca. 8. Transfer to 1 L Wheaton bottles.
Ingredient for 1 L to 4 L.
Reagent
1 Liter
trizma (tris) base
108 grams
boric acid
55 grams
EDTA-Disodium salt 9.3 grams
2 Liter
3 Liter
4 Liter
216 grams
110 grams
18.6 grams
324 grams
165 grams
27.9 grams
432 grams
220 grams
37.2 grams
5
Department of Molecular Biology
Methods Handbook
B. Electrophoresis Buffers
3. Tris-Borate (TBE) Buffer - LOW STRENGTH 10x Stock
Follow same instructions as above but with reduced amounts of
ingredients shown below.
Reagent
1 Liter
trizma (tris) base
60.6 grams
boric acid
30.9 grams
EDTA-Disodium salt 3.4 grams
2 Liter
3 Liter
4 Liter
121.2 grams
61.8 grams
6.8 grams
181.8 grams
92.7 grams
10.2 grams
242.4 grams
123.6 grams
13.6 grams
6
Department of Molecular Biology
Methods Handbook
C. Liquid Media
1. LB (Luria-Bertani) Medium
A. FROM SCRATCH
Weigh out the amounts of the first three ingredients (i.e. the solid
ingredients) listed below that corresponds to the final volume of media into a volumetric
beaker of the appropriate size. Add about 3/4 of the final solution volume of pure water and
stir until dissolved using a stirring bar. Next, pipette the amount of 2N NaOH called for in
the table into the solution. Add enough pure water to bring the solution up to the desired
volume and stir.
Transfer the media in 100ml aliquots to whichever size screw top bottles
are required. Screw on caps hand tight, and then loosen one turn. Place in autoclave
for 30 minutes at 250° F, 20 psi on liquid cycle.
Reagent
1 Liter
bacto-tryptone
10 grams
bacto-yeast extract 5 grams
NaCl, crystalline
5 grams
2 Liter
3 Liter
4 Liter
20 grams
10 grams
10 grams
30 grams
15 grams
15 grams
40 grams
20 grams
20 grams
B. FROM GIBCO LB BROTH BASE
Weigh out 80 grams of American Biorganics, Inc. LB Broth Base (cat. # A12-5150)
into a 4-liter plastic beaker. Add 4 liters of pure water and stir with a stirring bar until
dissolved. Transfer media to appropriate size bottles and autoclave according
to instruction above. (Amounts other than 4 liters can be made; simply weigh out
20 grams of the broth base per liter of stock.)
7
Department of Molecular Biology
Methods Handbook
C. Liquid Media
2. M9 Salts for Medium (10x Salt Stock)
A. FROM SCRATCH
Weigh out the amounts of the four salts shown below according to the
final volume of stock solution you wish to make into an appropriately sized
volumetric beaker. Add about 3/4 of the final solution volume of pure water and
stir using a stirring bar until dissolved. Dilute up to final volume with pure water
and stir further.
Transfer the salt solution in 100ml aliquots to the desired size screw top
bottles. Screw on caps hand tight, and then loosen one turn. Place in
autoclave for 30 minutes at 250° F, 20 psi on liquid cycle.
Ingredient for 1L for 2L
Reagent
1 Liter
2 Liter
3 Liter
4 Liter
Na2HPO4 (Dibasic Sodium Phosphate)
58 grams
30 grams
5 grams
116 grams
60 grams
10 grams
174 grams
90 grams
15 grams
232 grams
120 grams
20 grams
KH2PO4 (Monobasic potassium phosphate)
NaCl (Crystalline)
B. FROM GIBCO M-9 MINIMAL SALTS MIX
Weigh out 100 grams of Gibco M-9 Minimal Salts mix (cat. # 12980-041) for
each liter of media you wish to make into an appropriately sized plastic beaker.
Add the required amount of pure water and stir with a stirring bar until dissolved.
Transfer media to appropriate size bottles and autoclave according to
instruction above. NOTE: THIS MIX DOES NOT CONTAIN ANY SODIUM
CHLORIDE (NaCl).
8
Department of Molecular Biology
Methods Handbook
C. Liquid Media
3. TY Medium
Weigh out the amounts of the ingredients listed below corresponding to
the final volume of media you wish to make into a volumetric beaker of the
appropriate size. Add about 3/4 of the final solution volume of pure water and
stir until dissolved using a stirring bar.
Transfer the media in 100ml aliquots to whichever size screw top bottles
are required. Screw on caps hand tight, and then loosen one turn. Place in autoclave
for 25 minutes at 250° F, 20 psi on liquid cycle. REMOVE FROM AUTOCLAVE
IMMEDIATELY.
Reagent
1 Liter
bacto-tryptone
5 grams
bacto-yeast extract 3 grams
CaCl2.2H2O
0.5 grams
2 Liter
3 Liter
4 Liter
10 grams
6 grams
1.0 grams
15 grams
9 grams
1.5 grams
20 grams
12 grams
2.0 grams
9
Department of Molecular Biology
Methods Handbook
C. Liquid Media
4. YPD Medium
A. FROM SCRATCH
Weigh out the amounts of the ingredients listed below corresponding to
the final volume of media you wish to make into a volumetric beaker of the
appropriate size. Add about 3/4 of the final solution volume of pure water and
stir until dissolved using a stirring bar. Transfer the media in 100ml aliquots to whichever
size screw top bottles are required. Screw on caps hand tight, and then loosen one turn.
Place in autoclave for 25 minutes at 250° F, 20 psi on liquid cycle. REMOVE FROM
AUTOCLAVE IMMEDIATELY.
Reagent
1 Liter
dextrose, anhydrous 20 grams
bacto-peptone
20 grams
bacto-yeast extract 10 grams
2 Liter
3 Liter
4 Liter
40 grams
40 grams
20 grams
60 grams
60 grams
30 grams
80 grams
80 grams
40 grams
B. FROM GIBCO YEPD BROTH
Weigh out 50 grams of American Bioanalytical YEPD Broth (cat. # 7295) for each
liter of media you wish to make into an appropriately sized plastic beaker. Add the
required amount of pure water and stir with a stirring bar until dissolved.
Transfer media to appropriate size bottles and autoclave according to
instruction above.
10
Department of Molecular Biology
Methods Handbook
D. Plates
1. LB (Luria-Bertani) Plates
A. FROM SCRATCH
Weigh out the amounts of the first four ingredients (i.e. the solid
ingredients) listed below corresponding to the final volume of plate media you
wish to make into a volumetric flask of the appropriate size. Typically, 1 liter of
plate media is made in a 2800ml wide mouth fernbach flask. Add about 3/4 of
the final solution volume of pure water and pipette in the indicated amount of 2N
NaOH. Stir manually by swirling gently until dissolved. Add enough pure water
to bring the solution up to the desired volume and stir again by swirling gently.
Place foil over the top of the flask and autoclave for 30 minutes at 250° F, 20 psi
on liquid cycle. Remove, allow the broth to cool somewhat, but pour into Petri
dishes while still warm.
Reagent
1 Liter
2 Liter
3 Liter
4 Liter
bacto-tryptone
bacto-yeast extract
NaCl
bacto-agar
2 N NaOH
10 grams
5 grams
5 grams
15 grams
1.5 mL
20 grams
10 grams
10 grams
30 grams
3.0 mL
30 grams
15 grams
15 grams
45 grams
4.5 mL
40 grams
20 grams
20 grams
50 grams
6.0 mL
B. FROM GIBCO LB AGAR
Weigh out into an appropriately sized flask 32 grams of Gibco LB Agar
(cat. # M27000) per liter of plate media you wish to make. Add the correct
amount of pure water and stir using a stirring bar until dissolved. Add one drop
of antifoaming agent. Place foil over the flask and follow the autoclaving
instructions above.
11
Department of Molecular Biology
Methods Handbook
D. Plates
2. TYPlates
Weigh out the amounts of the four ingredients listed below corresponding
to the final volume of plate media you wish to make into a volumetric flask of the
appropriate size. Typically, 1 liter of plate media is made in a 2800ml wide
mouth fernbach flask. Add about 3/4 of the final solution volume of pure water.
Stir manually by swirling gently until dissolved. Add enough pure water to bring
the solution up to the desired volume and stir again by swirling gently. Place foil
over the top of the flask and autoclave for 30 minutes at 250° F, 20 psi on liquid
cycle. Remove immediately from autoclave, allow the broth to cool
somewhat, but pour into Petri dishes while still warm.
Reagent
1 Liter
2 Liter
3 Liter
4 Liter
Bacto-tryptone
bacto-yeast extract
calcium chloride
bacto-agar
5 grams
3 grams
0.5 grams
15 grams
10 grams
6 grams
1.0 grams
30 grams
15 grams
9 grams
1.5 grams
45 grams
20 grams
12 grams
2.0 grams
60 grams
12
Department of Molecular Biology
Methods Handbook
D. Plates
3. YPDPlates
A. FROM SCRATCH
Weigh out the amounts of the four ingredients listed below corresponding
to the final volume of plate media you wish to make into a volumetric flask of the
appropriate size. Typically, 1 liter of plate media is made in a 2800ml wide
mouth fernbach flask. Add exactly half of the final solution volume of pure water.
Stir manually by swirling gently until dissolved. Add the remaining half of pure
water to bring the solution up to the desired volume and stir again by swirling
gently. Place foil over the top of the flask and autoclave for 25 minutes at 250° F,
20 psi on liquid cycle. Remove from autoclave, allow the broth to cool
somewhat, but pour into Petri dishes while still warm.
Reagent
bacto-yeast extract
bacto-peptone
dextrose
bacto-agar
1 Liter
10 grams
20 grams
20 grams
20 grams
2 Liter
20 grams
40 grams
40 grams
40 grams
3 Liter
30 grams
60 grams
60 grams
60 grams
4 Liter
40 grams
80 grams
80 grams
80 grams
B. FROM GIBCO YEPD AGAR
Weigh out into an appropriately sized flask 66 grams of Gibco YEPD
Agar (cat. # M56800) per liter of plate media you wish to make. Add the correct
amount of pure water and stir using a stirring bar until dissolved. Add one drop
of antifoam agent. Place foil over the flask and follow the autoclaving
instructions above.
13
Department of Molecular Biology
Methods Handbook
D. Plates
4. RNAi Plates
Reagent
NaCl
Agar
Bactopeptone
1 Liter
3 grams
17 grams
2.5 grams
2 Liter
6 grams
34 grams
5 grams
3 Liter
9 grams
51 grams
7.5 grams
4 Liter
12 grams
68 grams
10 grams
Weigh out the amounts of the three ingredients listed above corresponding
to the final volume of plate media you wish to make into a volumetric flask of the
appropriate size. Typically, 1 liter of plate media is made in a 2800ml wide
mouth fernbach flask. Add exactly half of the final solution volume of pure water.
Stir manually by swirling gently until dissolved. Add the remaining half of pure
water to bring the solution up to the desired volume and stir again by swirling
gently. Place foil over the top of the flask and autoclave for 25 minutes at 250° F,
20 psi on liquid cycle. Remove from autoclave, and add the following chemicals in the
appropriate amount.
Reagent
1 Liter
1M Magnesum Sulfate
1 mL
5 mg/mL Cholesterol
1 mL
1 M Potassium Monophosphate 25 mL
2 Liter
2 mL
2 mL
50 mL
3 Liter
3 mL
3 mL
75 mL
4 Liter
4 mL
4 mL
100 mL
Allow to cool to 60°C then add the following chemicals in the appropriate
amount.
Reagent
1 M Calcium Chloride
25 mg/mL Carbenicillin
0.2 g/mL IPTG (Frozen stock)
1 Liter
1 mL
1 mL
6 mL
2 Liter
2 mL
2 mL
12 mL
14
3 Liter
3 mL
3 mL
18 mL
4 Liter
4 mL
4 mL
24 mL
Department of Molecular Biology
Methods Handbook
D. Plates
5. Standard Worm Plates
Take 1100 grams of Bacto-agar, 150 grams of NaCl, and 125 grams of
bactopeptone in a plastic bottle and mix the three powders well by shaking.
Reagent
Above Powder
1 Liter
25.5 grams
2 Liter
51 grams
3 Liter
76.5 grams
4 Liter
102 grams
Weigh out the amount of the powder listed above corresponding
to the final volume of plate media you wish to make into a volumetric flask of the
appropriate size. Typically, 1 liter of plate media is made in a 2800ml wide
mouth fernbach flask. Add exactly half of the final solution volume of pure water.
Stir manually by swirling gently until dissolved. Add the remaining half of pure
water to bring the solution up to the desired volume and stir again by swirling
gently. Place foil over the top of the flask and autoclave for 40 minutes at 250° F,
20 psi on liquid cycle. Remove from autoclave, and immediately add the following
chemicals in the appropriate amount.
Reagent
1 Liter
1M Magnesum Sulfate
1 mL
5 mg/mL Cholesterol
1 mL
1 M Potassium Monophosphate 25 mL
1% Nystatin
1 mL
2 Liter
2 mL
2 mL
50 mL
2 mL
3 Liter
3 mL
3 mL
75 mL
3 mL
4 Liter
4 mL
4 mL
100 mL
4 mL
Place in cold-room to cool to 55° C then add the following chemicals in the
appropriate amount. Stir until dissolved, pour the plates while the solution is still liquid.
Reagent
1 M Calcium Chloride
2.5% Streptomycin
1 Liter
1 mL
7.5 mL
2 Liter
2 mL
15 mL
15
3 Liter
3 mL
22.5 mL
4 Liter
4 mL
30 mL
Department of Molecular Biology
Methods Handbook
D. Plates
5. Enriched Worm Plates
Weigh out the amount of the powder listed below corresponding
to the final volume of plate media you wish to make into a volumetric flask of the
appropriate size. Typically, 1 liter of plate media is made in a 2800ml wide
mouth fernbach flask. Add exactly half of the final solution volume of pure water.
Stir manually by swirling gently until dissolved. Add the remaining half of pure
water to bring the solution up to the desired volume and stir again by swirling
gently. Place foil over the top of the flask and autoclave for 40 minutes at 250° F,
20 psi on liquid cycle. Remove from autoclave, and immediately add the following
chemicals in the appropriate amount.
Reagent
Agar
NaCl
Bactopeptone
1 Liter
25 grams
1.2 grams
20 grams
2 Liter
50 grams
2.4 grams
40 grams
3 Liter
75 grams
3.6 grams
60 grams
4 Liter
100 grams
4.8 grams
80 grams
Place in cold-room to cool to 55° C then add the following chemicals in the
appropriate amount. Stir until completely dissolved, pour the plates while the solution is
still liquid.
Reagent
1 M Calcium Chloride
5 mg/ml Cholesterol
1 M Potassium Phosphate
1 Liter
1 mL
1 mL
1 mL
2 Liter
2 mL
2 mL
2 mL
16
3 Liter
3 mL
3 mL
3 mL
4 Liter
4 mL
4 mL
4 mL
Department of Molecular Biology
Methods Handbook
E. Antibiotic Plates
1. Ampicillin (Amp) Plates
Ampicillin stock is 5 mg/ml, made as follows: weigh out 0.5 grams of
powdered ampicillin into a flask and add 75 mLs of pure water. Stir until
completely dissolved then transfer to a 100 ml volumetric flask and fill to 100
mLs. Working in an Edgegard hood, sterilize by filtering through a Nalgene filter
into a screw-top 100ml sterile bottle. Label ten tubes with the antibiotic
name, concentration, date of preparation, and the preparer's initials. Sterilely dispense 10
mL aliquots of stock into sterile tubes, and then store in the freezer for up to 10 days. Thaw
prior to use in plates.
Ampicillin plates are 50 ug/ml, made as follows: Allow the autoclaved
LB media to cool to ca. 48° C. Add 10 mLs of the frozen ampicillin stock per liter of
media and mix by hand slowly, avoiding the formation of air bubbles. Pour
plates immediately thereafter. Amp plates may be stored at 4° C for a maximum
of two weeks before use.
Amp/TET OMNI square plates are poured using a 4-liter mix, which is made up
as follows. 128 grams of LB agar, 6 mLs of 2N NaOH, and 4 drops of Antifoam A are
added to one liter of water. The water is then made up to a total volume of 4 liters, then
autoclaved for 30 minutes. Allow to cool to ca. 48° C. Add 12 mLs of 5 mg/ml TET
solution, and then add 2 mLs of 100 mg/ml AMP solution. Pour plates, using 50 mLs of
mix per plate.
17
Department of Molecular Biology
Methods Handbook
E. Antibiotic Plates
2. Chloramphenicol (Cam) Plates
Chloramphenicol stock is 100 mg/ml, made as follows: weigh out
exactly 10 grams of crystalline chloramphenicol into a flask and add 75 mLs of
absolute ethanol. Stir until completely dissolved then transfer to a 100 ml
volumetric flask, which was pre-rinsed with absolute ethanol, and fill to 100 mLs
with absolute ethanol. Label ten tubes with the antibiotic name, concentration, date of
preparation, and the preparer's initials. Sterilely dispense 10 mL aliquots of stock into
sterile tubes, and then store in the freezer for up to 4 months. Thaw prior to use in plates.
Chloramphenicol plates are 30 ug/ml, made as follows: Allow the
autoclaved LB media to cool to ca. 48° C. Add 0.3 mLs of the chloramphenicol
stock per liter of media and mix by hand slowly, avoiding the formation of air
bubbles. Pour plates immediately thereafter. Cam plates may be stored at 4° C
and should be used within 5 days.
18
Department of Molecular Biology
Methods Handbook
E. Antibiotic Plates
3. Kanamycin (Kan) Plates
Kanamycin stock is 10 mg/ml, made as follows: weigh out 1.0 gm of
solid kanamycin acid sulfate into a flask and add 75 mLs of pure water. Stir until
completely dissolved then transfer to a 100 ml volumetric flask and fill to 100
mLs. Working in an Edgegard hood, sterilize by filtering through a Nalgene filter
into a screw-top 100ml sterile bottle. Label fifty tubes with the antibiotic
name, concentration, date of preparation, and the preparer's initials. Sterilely dispense 2 mL
aliquots of stock into sterile tubes, and then store in the freezer for a maximum of 10 days.
Thaw prior to use in plates.
Kanamycin plates are 20 ug/ml, made as follows: Allow the
autoclaved LB media to cool to ca. 48° C. Add 2.0 mLs of the kanamycin stock
per liter of media and mix by hand slowly, avoiding the formation of air bubbles.
Pour plates immediately thereafter. Kan plates may be stored at 4° C for a
maximum of four weeks before use.
19
Department of Molecular Biology
Methods Handbook
E. Antibiotic Plates
4. Neomycin (Nm) Plates
Neomycin stock is 5 mg/ml, made as follows: weigh out 0.5 gm of solid
neomycin sulfate into a flask and add 75 mLs of pure water. Stir until completely
dissolved then transfer to a 100 ml volumetric flask and fill to 100 mLs. Working
in an Edgegard hood, sterilize by filtering through a Nalgene filter into a 100 ml
screw-top sterile bottle. Label twenty tubes with the antibiotic name, concentration, date of
preparation, and the preparer's initials. Sterilely dispense 5 mL aliquots of stock into sterile
tubes, and then store in the freezer for up to a month. Thaw prior to use in plates.
Neomycin plates are 25 ug/ml, made as follows: Allow the autoclaved
LB media to cool to ca. 48° C. Add 5.0 mLs of the neomycin stock per liter of
media and mix by hand slowly, avoiding the formation of air bubbles. Pour
plates immediately thereafter. Nm plates may be stored at 4° C for a maximum of
5 weeks before use.
20
Department of Molecular Biology
Methods Handbook
E. Antibiotic Plates
5. Streptomycin (Sm) Plates
Streptomycin stock is 50 mg/ml, made as follows: weigh out 5.0 gm of
solid streptomycin into a flask and add 75 mLs of pure water. Stir until
completely dissolved then transfer to a 100 ml volumetric flask and fill to 100
mLs. Working in an Edgegard hood, sterilize by filtering through a Nalgene filter
into a 100 ml screw-top sterile bottle. Label ten tubes with the antibiotic
name, concentration, date of preparation, and the preparer's initials. Sterilely dispense 10
mL aliquots of stock into sterile tubes, and then store in the freezer. Thaw prior to use in
plates.
Streptomycin plates are 500 ug/ml, made as follows: Allow the
autoclaved LB media to cool to ca. 48° C. Add 10.0 mLs of the streptomycin stock
per liter of media and mix by hand slowly, avoiding the formation of air bubbles.
Pour plates immediately thereafter. Sm plates may be stored at 4° C for a
maximum of 5 weeks before use.
21
Department of Molecular Biology
Methods Handbook
E. Antibiotic Plates
6. Tetracycline (Tet) Plates
Tetracycline stock is 1 mg/ml, made as follows: weigh out 0.1 gm of
solid tetracycline hydrochloride into a flask and add 75 mLs of pure water. Stir
until completely dissolved then transfer to a 100 ml volumetric flask and fill to
100 mLs. Working in an Edgegard hood, sterilize by filtering through a Nalgene
filter into a 100 ml screw-top sterile bottle. Label ten tubes with the antibiotic
name, concentration, date of preparation, and the preparer's initials. Sterilely dispense 10
mL aliquots of stock into sterile tubes, and then store in the freezer. Thaw prior to use in
plates.
Tetracycline plates are 10 ug/ml, made as follows: Allow the
autoclaved LB media to cool to ca. 48° C. Add 10.0 mLs of the tetracycline stock
per liter of media and mix by hand slowly, avoiding the formation of air bubbles.
Pour plates immediately thereafter. Tet plates may be stored at 4° C in the dark
for a maximum of 2 weeks before use.
22
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
1. 20% Ammonium Sulfate (20% (NH4)2 SO4)
Weigh out 200.0 grams of solid ammonium sulfate into a large beaker or
flask and add 750 mLs of pure water. Stir with stirring bar until dissolved. Add an
additional 250 mLs of pure water and stir again briefly.
Transfer 100 ml aliquots to the square 'milk' bottles. Screw on caps hand
tight and then loosen one turn. Place in the autoclave and sterilize for 30
minutes at 250° F, 20 psi on liquid cycle. Label bottles with name and
concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
2. 10x BS Stock Solution
Add 1600 mLs of pure water into a 2800 ml fernbach flask. Weigh out 90.0
grams of solid potassium phosphate monobasic ( KH2 PO4 ). Add this to the flask
and stir with a stirring bar and heat gently until the salt is dissolved. Weigh out
210.0 grams of potassium phosphate dibasic (K2 HPO4) into the solution and stir
and heat until it is dissolved. Weigh out 20.0 grams of ammonium sulfate
((NH4)2SO4 ) and add it to the solution. Weigh out 1.0 gm of magnesium
sulfate hepta-hydrate (MgSO4.7H2O) and add it to the solution. Next, pipette in 0.035ml
of stock solution 5M NaCl, and 0.07 ml of stock solution 1M CaCl2. Stir until all
solids are dissolved and transfer to a two liter graduated cylinder and add
enough pure water to bring the volume up to two liters.
Transfer 100 ml aliquots to the square 'milk' bottles. Screw on caps hand
tight and then loosen one turn. Place in the autoclave and sterilize for 30
minutes at 250° F, 20 psi on liquid cycle. Label bottles with name and
concentration of chemical, date prepared, and preparer's initials.
23
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
3. 1M Calcium Chloride (1M CaCl2)
Dissolve 147.0 grams of CaCl2.2H2 O into 800 mLs of pure water. Transfer
solution to a one liter graduated cylinder and bring volume up to one liter with
pure water. Dispense into aliquots and bottles of desired amounts and sizes. Screw
on caps hand tight and then loosen one turn. Place in the autoclave and
sterilize for 30 minutes at 250° F, 20 psi on liquid cycle. Label bottles with name
and concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
4. 20% CAS Amino Acids
With the aid of a stirring bar, dissolve 200.0 grams of CAS amino acids into
800 mLs of pure water in a one or two liter flask. Transfer solution to a one-liter
graduated cylinder and bring volume up to one liter with pure water. Dispense into 100 ml
aliquots in round bottles. Screw on caps hand tight and then loosen one turn. Place in the
autoclave and sterilize for 30 minutes at 250° F, 20 psi on liquid cycle. Label bottles with
name and concentration of chemical, date prepared, and preparer's initials.
24
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
5. 40% Glucose
With the aid of a stirring bar/heating plate, dissolve 400.0 grams of glucose
(anhydrous dextrose) into 600 mLs of pure water in a one or two liter flask. Heat
the solution gently to aid dissolution. Transfer solution to a one-liter graduated
cylinder and bring volume up to one liter with pure water.
Dispense into 100 ml aliquots in round bottles. Screw on
caps hand tight and then loosen one turn. Place in the autoclave and sterilize
for 20 minutes (no longer!) at 250° F, 20 psi on liquid cycle. Remove from
autoclave immediately to prevent crystallization. Label bottles with name and
concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
6. 10x Nif-P Stock Solution
Weigh out 2.0 gm of solid MgSO4.7H2O into a 2 liter or fernbach flask.
Pipette in 0.07 mLs of 1M CaCl2 stock solution and 0.035 mLs of 5M NaCl stock
solution into the flask. Pipette in 10.0 mLs of Na2MoO4 (sodium molybdate) stock
solution (see page 27). Add 990 mLs of pure water and stir until dissolved and
thoroughly mixed.
Dispense into 100 ml aliquots in square bottom 'milk' bottles. Screw on
caps hand tight and then loosen one turn. Place in the autoclave and sterilize
for 30 minutes at 250° F, 20 psi on liquid cycle. Label bottles with name and
concentration of chemical, date prepared, and preparer's initials.
25
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
7. 10x Phosphate (PO4) Stock Solution
Weigh out 696.8 grams of K2HPO4 (potassium phosphate dibasic) into a
2800 ml fernbach flask. Dilute up to 2 liters with pure water. Use a stirring
bar/heating plate to dissolve.
Into a separate 2800 ml fernbach flask, weigh out 544.4 grams of KH2PO4
(potassium phosphate monobasic). Dilute up to 2 liters with pure water. Use a
stirring bar/heating plate to dissolve and prevent precipitation. Measure out 380 mLs of
the monobasic solution and 1,620 mLs of the dibasic solution into a 4-liter flask. Add
2000 mLs of pure water.
Dispense into 100 ml aliquots in square bottom 'milk' bottles. Screw on
caps hand tight and then loosen one turn. Place in the autoclave and sterilize
for 30 minutes at 250° F, 20 psi on liquid cycle. Label bottles with name and
concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
8. 40% Sucrose
With the aid of a stirring bar/heating plate, dissolve 400.0 grams of sucrose
into 800 mLs of pure water in a one or two liter flask. Heat the solution gently to
aid dissolution. Transfer solution to a one liter graduated cylinder and bring
volume up to one liter with pure water.
Dispense into 100 ml aliquots in round bottles. Screw on
caps hand tight and then loosen one turn. Place in the autoclave and sterilize
for 20 minutes (no longer!) at 250° F, 20 psi on liquid cycle. Remove from
autoclave immediately to prevent crystallization. Label bottles with name and
concentration of chemical, date prepared, and preparer's initials.
26
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
9. 5M Sodium Chloride (5M NaCl)
Dispense into aliquots and bottles of desired amounts and sizes. Screw
on caps hand tight and then loosen one turn. Place in the autoclave and
sterilize for 30 minutes at 250° F, 20 psi on liquid cycle. Label bottles with name
and concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
10. 2N Sodium Hydroxide (2N NaOH)
Weigh out into a 250 ml flask 8.0 grams of NaOH pellets. Add 80 mLs of
pure water and stir until dissolved. Pour into a graduated cylinder and bring
volume up to 100 mLs with pure water. Transfer to a 100 ml screw top bottle. Label the
bottle with name and concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
11. Sodium Molybdate (Na2MoO4) Stock Solution
Weigh out 5.0 grams of Na2MoO4.2H2O solid into a 2-liter flask. Add 1 liter
of pure water and stir until dissolved. Dispense into aliquots and bottles of desired amounts
and sizes. Screw on caps hand tight and then loosen one turn. Place in the autoclave and
sterilize for 30 minutes at 250° F, 20 psi on liquid cycle. Label bottles with name
and concentration of chemical, date prepared, and preparer's initials.
27
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
12. Water(Sterile)
Fill a 500 ml Wheaton screw-top bottle with 425 mLs of pure water (or fill
other bottles as required). Screw on cap hand tight and then loosen one turn.
BE SURE not to use a cap with a worn or missing seal. Place in the autoclave
and sterilize for 30 minutes at 250° F, 20 psi on liquid cycle. After removing from
the autoclave, tighten the cap as soon as the bottle is cool enough to touch
(within 1-2 minutes). Do not transport the bottle until the cap has been
tightened.
F. Stock Solutions
13. 1 M Magnesium Sulfate.
Weigh out into a 1 L flask 246.5 grams of Magnesium Sulfate. Add 800 mLs of
pure water and stir until dissolved. Pour into a volumetric flask and bring
volume up to 1000 mLs with pure water. Transfer to a screw top bottle. Autoclave for 30
minutes on the liquid cycle. Label the bottle with name and concentration of chemical,
date prepared, and preparer's initials.
Reagent
1M Magnesum Sulfate
1 Liter
246.5 grams
2 Liter
493 grams
3 Liter
739.5 grams
4 Liter
986 grams
F. Stock Solutions
14. 1 M Calcium Chloride.
Weigh out into a 1 L flask 147 grams of Calcium Chloride. Add 800 mLs of
pure water and stir until dissolved. Pour into a volumetric flask and bring
volume up to 1000 mLs with pure water. Transfer to a screw top bottle. Autoclave for 30
minutes on the liquid cycle. Label the bottle with name and concentration of chemical,
date prepared, and preparer's initials.
28
Department of Molecular Biology
Methods Handbook
Reagent
1 M Calcium Chloride
1 Liter
147 grams
2 Liter
296 grams
3 Liter
443 grams
4 Liter
592 grams
F. Stock Solutions
15. 1 M Potassium Phosphate.
Weigh out into a 1 L flask 118.1 grams of KH2PO 4 and 30.1 grams of K2HPO 4.
Add 800 mLs of pure water and stir until dissolved. Pour into a volumetric flask and
bring volume up to 1000 mLs with pure water. Transfer to a screw top bottle. Autoclave
for 30 minutes on the liquid cycle. Label the bottle with name and concentration of
chemical, date prepared, and preparer's initials.
Reagent
1 M Potassium Monobasic
1 M Potassium Dibasic
1 Liter
118.1 grams
30.1 grams
2 Liter
236.2 grams
60.2 grams
3 Liter
354.3 grams
90.3 grams
4 Liter
472.4 grams
120.4 grams
F. Stock Solutions
16. 5 mg/mL cholesterol.
Weigh out into a 100 mL flask 0.5 grams of Cholesterol. Add 80 mL of ethanol
and stir until dissolved. Pour into a volumetric flask and bring volume up to 100 mLs
with ethanol. Transfer to a screw top bottle. DO NOT autoclave. Label the bottle with
name and concentration of chemical, date prepared, and preparer's initials.
F. Stock Solutions
17. S Basal Solution.
Weigh out into an appropriately sized flask 23.6 grams of NaCl and 200 mLs of
1 M Potassium Phosphate. Add 1 L of water and stir until dissolved. Pour into a
volumetric flask and bring volume up to 4 liters with water. Transfer to a screw top
bottle. Autoclave for 30 minutes on the liquid cycle. Label the bottle with name and
concentration of chemical, date prepared, and preparer's initials.
29
Department of Molecular Biology
Methods Handbook
F. Stock Solutions
18. Streptomycin.
Weigh out into a 1 L flask 25 grams of Streptomycin. Add 800 mL of water and
stir until dissolved. Pour into a volumetric flask and bring volume up to 1 liter with
water. Sterilize using a filter and then transfer to a screw top bottle. Label the bottle
with name and concentration of chemical, date prepared, and preparer's initials, and store
at 4°C.
F. Stock Solutions
19. Nystatin.
Weigh out into a 100 mL flask 1 grams of Nystatin. Add 70 mL of Ethanol and
stir until dissolved. Pour into a volumetric flask and bring volume up to 100 mL with
water. DO NOT autoclave. Transfer into a screw top bottle, and wrap the bottle in foil.
Label the bottle with name and concentration of chemical, date prepared, and preparer's
initials, and store at 4°C in the dark.
F. Stock Solutions.
20. M9 Buffer.
Weigh out into an appropriately sized flask take 12 grams of KH2PO 4, 24 grams
of Na2 HPO 4, and 20 grams of NaCl. Add 1 L of water and stir until dissolved. Pour into
a volumetric flask and bring volume up to 4 liters with water. Transfer to a screw top
bottle. Autoclave for 30 minutes on the liquid cycle. Allow to cool and then add 4 mL of
1 M MgSO4. Label the bottle with name and concentration of chemical, date prepared,
and preparer's initials.
30
Department of Molecular Biology
Methods Handbook
F. Stock Solutions.
14. OP50 for worm plates.
Take 4 bottles of sterile LB from chemical room. Flame the lid prior to opening it.
Add 5 mL of streptomycin solution to 500 mL of sterile Luria Broth. Flame the loop
thoroughly until it turns red. Allow it to cool down, touch it to the agar, and then pick a
single colony from the OP50 plate. Place the loop with bacteria on it into the LB bottle.
Flame the loop once again and then repeat. Four bottles is sufficient for one week.
Incubate overnight in a 37° C incubator. Store in a 4° C refrigerator.
31