Recipes for Reagents and Stock Solutions
The success of the laboratories depends on the use of high-quality reagents. Follow the recipes with care
and pay attention to cleanliness. Use a clean spatula for each ingredient or carefully pour each ingredient
from its bottle.
I. DNA Isolation
4.4 M Ammonium Acetate (NH4OAc)
Edward's Buffer
0.5 M Ethylene Diamine Tetraacetic Acid (EDTA)
Phenol:chloroform (2:1; volume:volume)
5 mg/ml RNase A (Pancreatic RNase)
4 M Sodium Chloride (NaCl)
10% Sodium Dodecyl Sulfate (SDS)
1 M Tris (pH 8.0)
Tris/EDTA (TE) Buffer
Tris/EDTA Buffer with RNase A (TER)
Urea Extraction Buffer
II. Polymerase Chain Reaction
1% Cresol Red Dye
Cresol Red Loading Dye
KOD Hot Start DNA Polymerase
Primer/Loading Dye Mix
Primer Sequences
III. Agarose Gel Electrophoresis
1.0 or 2.0% Agarose
1 µg/ml Ethidium Bromide Staining Solution
0.2% Methylene Blue Stock Solution
0.025% Methylene Blue Staining Solution
pBR322/
BstNI Size Markers (0.1 µg/µl)
100 bp or 1 kb DNA Ladder (0.125 µg/µl)
20X Tris/Borate/EDTA (TBE) Electrophoresis Buffer
1X Tris/Borate/EDTA (TBE) Electrophoresis Buffer
Notes on Buffers
1. Typically, solid reagents are dissolved in a volume of deionized or distilled water equivalent to 70-
80% of the finished volume of buffer. This leaves room for the addition of acid or base to adjust the
pH. Then, water is added to bring the solution up to the final volume.
2. Buffers typically are used as 1X or 10X solutions. Buffers are diluted when mixed with other reagents
to produce a working concentration of 1X.
3. The commercial enzymes used for these laboratories all come from the suppliers with appropriate
buffers. These should be used unless noted otherwise.
4. Storage temperatures of 4°C and -20°C refer to normal refrigerator and freezer (non-frost free)
temperatures, respectively.
Recipes for Reagents and Stock Solutions
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I. DNA Isolation
4.4 M Ammonium Acetate (NH4OAc)
Makes approximately 200 ml.
Store at room temperature (indefinitely).
Mix in a 500-ml beaker in a fume hood:
50.5 ml glacial acetic acid
105 ml of deionized or distilled water
45 ml of ammonium hydroxide (add slowly)
Edward’s Buffer
Makes 50 ml.
Store at room temperature (indefinitely).
Mix in a 50-ml tube:
32.5 ml of deionized or distilled water
10 ml of 1 M Tris pH 8.0
2.5 ml of 5 M NaCl
2.5 ml of 0.5 M EDTA
2.5 ml of 10% SDS
0.5 M Ethylene Diamine Tetraacetic Acid (EDTA) (pH 8.0)
Makes 100 ml.
Store at room temperature (indefinitely).
1. Add 18.6 g EDTA (disodium salt, m.w. 372.24) to 80 ml deionized or distilled water.
2. Adjust to pH by slowly adding approximately 2.2 g of sodium hydroxide pellets (m.w. 40.00). (If a
pH meter is not available, adding 2.2 g of NaOH pellets will make a solution of approximately pH
8.0).
3. Mix vigorously with a magnetic stirrer or by hand. EDTA will only dissolve after pH has reached
8.0 or higher.
NOTE: Use only the disodium salt of EDTA.
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Copyright © 2005, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
CAUTION: Avoid inhaling acetic acid or ammonium hydroxide; wear goggles and work in a fume hood!
Phenol: chloroform (2:1; volume:volume)
Makes 60 ml.
Store at room temperature (indefinitely).
Mix in a 50-ml tube:
40 ml of phenol
20 ml of chloroform
5 mg/ml RNase A (Pancreatic RNase)
Makes 20 ml.
Store at –20°C (indefinitely).
1. Dissolve 100 mg of RNase A in 20 ml of 0.05% glacial acetic acid, and transfer to a 50-ml conical
tube.
2. Place the tube in a boiling-water bath for 15 minutes.
3. Cool the solution, and neutralize by adding 120 µl of 1 M Tris (pH 8.0).
4. Dispense 1-ml aliquots in 1.5-ml tubes.
NOTES:
• Use only RNase A from bovine pancreas.
• Dissolving RNase in the acetic acid prevents subsequent precipitation of the RNase. The solution
can be prepared by simply dissolving RNase in deionized or distilled water; however, the RNase
will occasionally precipitate from the solution and activity will be lost.
4 M Sodium Chloride (NaCl)
Makes 500 ml.
Store at room temperature (indefinitely).
1. Dissolve 116.9 g of NaCl (m.w. 58.44) in 250 ml of deionized or distilled water.
2. Add deionized or distilled water to make a total volume of 500 ml of solution.
Recipes for Reagents and Stock Solutions
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CAUTION: Avoid inhaling phenol or chloroform; wear goggles and work in a fume hood! Avoid skin contact with
phenol as it can cause severe burns.
CAUTION: Avoid inhaling acetic acid; wear goggles and work in a fume hood!
10% Sodium Dodecyl Sulfate (SDS)
Makes 100 ml.
Store at room temperature (indefinitely).
1. Dissolve 10 g electrophoresis-grade SDS (m.w. 288.37) in 80 ml deionized water.
2. Add deionized or distilled water to make 100 ml total solution.
NOTE: SDS is the same as sodium lauryl sulfate.
1 M Tris (pH 8.0)
Makes 100 ml.
Store at room temperature (indefinitely).
1. Dissolve 12.1 g Tris base (m.w. 121.10) in 70 ml deionized or distilled water.
2. Adjust pH by slowly adding concentrated hydrochloric acid (HCl); monitor with a pH meter.
3. Add deionized or distilled water to make 100 ml total solution.
NOTES:
• A yellow-colored solution indicates poor-quality Tris. Discard, and obtain from a different source.
• Many types of electrodes do not accurately measure the pH of Tris solutions; check with
manufacturer to obtain a suitable one.
• The pH of Tris solutions is temperature dependent; make pH measurements at room temperature.
Tris/EDTA (TE) Buffer
Makes 100 ml.
Store at room temperature (indefinitely).
Mix in a 200-ml beaker:
99 ml of deionized or distilled water
1 ml of 1 M Tris pH 8.0
200 µl of 0.5 M EDTA
Tris/EDTA Buffer with RNase A (TER)
Makes 5 ml.
Make fresh each time.
Mix:
100 µl 5 mg/ml RNase A
4.9 ml TE buffer
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Copyright © 2005, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
CAUTION: Avoid inhaling Tris powder; wear mask over nose and mouth!
CAUTION: Avoid inhaling SDS powder; wear mask over nose and mouth!
Urea Extraction Buffer
Makes 1000 ml.
Store at room temperature (indefinitely).
Mix in a 2000-ml beaker:
420 g of urea
87.5 ml of 4 M NaCl
50 ml of 1 M Tris pH 8.0
0.5 M EDTA
10 g n-lauryl sarcosine
Add deionized or distilled water to make a total volume of 1000 ml of solution.
II. Polymerase Chain Reaction
1% Cresol Red Dye
Makes 50 ml.
Store at room temperature (indefinitely).
Mix in a 50-ml tube:
500 mg cresol red dye
50 ml of distilled water
Cresol Red Loading Dye
Makes 50 ml.
Store at –20°C (indefinitely).
1. Dissolve 17 g of sucrose in 49 ml of distilled water in a 50-ml tube.
2. Add 1 ml of 1% cresol red dye and mix well.
KOD Hot Start DNA Polymerase and Buffer
KOD Hot Start DNA polymerase is purchased from Novagen (catalog number 71086-3). The
polymerase is supplied with 10X buffer, a 10X dNTP mix (2 mM each), and 25 mM MgSO4.
Recipes for Reagents and Stock Solutions
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Primer/Loading Dye Mixes (For Use with Ready-to-Go PCR Beads™ )
For
Arabidopsis Clf-2, CAPS markers,
Bz (Maize) analysis, and GMO testing
Makes enough for 50 reactions.
Store at –20°C for 1 year.
Mix in a 1.5-ml tube:
640 µl of distilled water
460 µl of Cresol Red Loading Dye
20 µl of 15 pmol/µl 5' primer
20 µl of 15 pmol/µl 3' primer
Primer Sequences
CLF genotyping:
primers for
CLF (Wt)
5'
CLF1
5'-TTAACCCGGACCCGCATTTGTTTCGG-3'
3'
CLF2
5'-AGAGAAGCTCAAACAAGCCATCGA-3'
primers for (Mutant)
5'
CLF1
5'-TTAACCCGGACCCGCATTTGTTTCGG-3'
3'
Ds
5'-GTCGGCGTGCGGCTGGCGGCG-3'
CAPS mapping:
primers for
m235-marker
m235-5’
5'-GAATCTGTTTCGCCTAACGC-3'
m235-3’
5'-AGTCCACAACAATTGCAGCC-3'
primers for
UFO-marker
UFO-5’
5'-GTGGCGGTTCAGACGGAGAGG-3'
UFO-3’
5'-AAGGCATCATGACTGTGGTTTTTC-3'
primers for g4026-marker
g4026-5’
5'-GGGGTCAGTTACATTACTAGC-3'
g4026-3’
5'-GTACGGTTCTTCTTCCCTTA-3'
primers
H77224-marker
H77224-5’
5’-GGATTTGGGGAAGAGGAAGTAA-3'
H77224-3’
5'-TCCTTAGCCTTGCTTTGATAGT-3'
Functional Genomics:
Example primers for control gene
At3g16240.1:
At3g16240.1 primer1
5’- gctcgatccacctaggctCAAGCATCTTCACAGGTTTTGG-3’
At3g16240.1 primer2
5'- cacagctccacctccacctccaggccggccAAATCAGCAGAAGCAAGAGGA-3'
At3g16240.1 primer3
5'- tgctggtgctgctgcggccgctggggccGTTCCTCTTGCTTCTGCTGATTTC-3'
At3g16240.1 primer4
5'- cgtagcgagaccacaggaCCATCCATTAATTTGTCATTGTTGT-3'
Find primers for
genes with unknown function at FTFLP
(http://aztec.stanford.edu/gfp/index.html). Use
Target Selection to
select a gene from a “short” list
of 4000 genes. Use
Search Database to
search for primer sequences for a locus. The course will
tag the locus
AT1G08480.
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Copyright © 2005, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
Bz (Maize) analysis:
primers for
bz (Wt)
bz-599
5'-CGAATGGCTGTTGCATTTCCAT-3'
bz-863R
5'-ACGGGACGCAGTTGGGCAGGA-3'
primers for
bz:
Ac
bz- 599
5'-CGAATGGCTGTTGCATTTCCAT-3'
Ac-132R
5'-TCTACCGTTTCCGTTTCCGTTT-3'
GMO testing:
primers for
35S-promoter
35S-5’
5’-CCGACAGTGGTCCCAAAGATGGAC-3’
35S-3’
5’-ATATAGAGGAAGGGTCTTGCGAAGG-3’
primers for tubulin
Tub5
5’- GGGATCCACTTCATGCTTTCGTCC-3'
Tub3
5'- GGGAACCACATCACCACGGTACAT-3’
III. Agarose Gel Electrophoresis
1.0 % or 2.0% Agarose
Makes 200 ml.
Use fresh or store solidified agarose at room temperature (several weeks).
Prepare a 2% agarose solution by adding 2 g of agarose to 100 ml of 1X TBE in a 500 ml flask or
beaker. Heat the flask or beaker in a boiling water bath (approximately 15 minutes) or in a microwave
oven (approximately 4 minutes) until the agarose is completely dissolved. You should no longer see
agarose particles floating in solution when the beaker is swirled. Allow the agarose to cool to approx-
imately 60°C, and hold at this temperature in a hot water bath. Cover beaker or flask with aluminum
foil, and skim any polymerized “skin” off the top of the solution before pouring.
NOTES:
• Samples of agarose powder can be preweighed and stored in capped test tubes until ready for
use.
• Solidified agarose can be stored at room temperature and then remelted over a boiling-water
bath (15-20 minutes) or in a microwave oven (3-5 minutes per beaker) prior to use. Always loosen
cap when remelting agarose in a bottle.
• When remelting agarose evaporation will cause the concentration to increase. If necessary,
compensate by adding back a small volume of water.
1 µ
g/ml Ethidium Bromide Staining Solution
Makes 500 ml.
Store in dark at room temperature (indefinitely).
1. Add 100 µl of 5 mg/ml ethidium bromide to 500 ml deionized or distilled water.
2. Store in unbreakable bottles (preferably opaque). Label bottles "CAUTION: Ethidium Bromide.
Mutagen and cancer-suspect agent. Wear rubber gloves when handling."
Recipes for Reagents and Stock Solutions
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NOTE: Ethidium bromide is light sensitive; store in dark container or wrap container in aluminum foil.
0.2% Methylene Blue Stock Solution
Makes 100 ml.
Store at room temperature (indefinitely).
1. Dissolve 0.2 g methylene blue-trihydrate (m.w. 373.9) in 100 ml of deionized or distilled water.
0.025% Methylene Blue Staining Solution
Makes 500 ml.
Store at room temperature (indefinitely).
1. Add 62.5 ml of 0.2% methelyene blue stock solution to 437.5 ml of deionized or distilled water.
pBR322/BstNI Size Markers (0.1 µ
g/µ
l)
Makes 100 µl.
Store at –20°C for 1 year.
Either buy pBR322 pre-cut with restriction enzyme
BstNI from New England Biolabs (#N3031) or
prepare it according to the following protocol:
1. Add 1 µl of a solution of 10 µg/µl pBR322 to 75 µl deionized or deionized or distilled water.
2. Add 10 µl 10x buffer (provided by supplier of the enzyme).
3. Add 5 µl
BstNI restriction enzyme, and incubate at 60°C for 60 min.
4. Electrophorese 5 µl (plus 1 µl loading dye) in a 1-2% agarose gel to check for complete digestion.
Exactly 5 bands should be visible, corresponding to 1,857 bp, 1,058 bp, 929 bp, 383, and 121 bp.
Any additional bands indicate incomplete digestion; add additional enzyme and incubate again
at 60°C.
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Copyright © 2005, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
CAUTION: Ethidium bromide is a mutagen by the Ames microsome assay and a suspected carcinogen. Wear
rubber gloves when preparing and using ethidium bromide solutions.
100 bp or 1 kb DNA Ladder (0.125 µ
g/µ
l)
Makes 100 µl.
Store at –20°C for 1 year.
Obtain the stock solution through New England Biolabs (#N3231S). The sold concentration is 0.5
µg/µl and should be kept at –20°C. You can dilute a small amount of the stock at a time following
this procedure:
1. Add 50 µl of deionized or distilled water to a 1.5-ml tube.
2. Add 25 µl of Cresol Red Loading Dye.
3. Add 25 µl of 100-bp or 1-kb DNA Ladder stock.
20X Tris/Borate/EDTA (TBE) Electrophoresis Buffer
Makes 1 liter.
Store at room temperature (indefinitely).
1. Add the following dry ingredients to 500 ml of deionized or distilled water in a 2-liter flask.
1 g of NaOH (m.w. 40.00)
216 g of Tris base (m.w. 121.10)
110 g of boric acid (m.w. 61.83)
14.8 g of EDTA (disodium salt, m.w. 372.24)
2. Stir to dissolve, preferably using a magnetic stir bar.
3. Add deionized or distilled water to bring total solution to 1 liter.
NOTE: If stored 20X TBE comes out of solution, place the flask in a water bath (37°C to 42°C) and stir
occasionally until all solid matter goes back into solution.
1X Tris/Borate/EDTA (TBE) Electrophoresis Buffer
Makes 10 liters.
Store at room temperature (indefinitely).
1. Into a spigoted carboy, add 9 liters deionized or distilled water to 1 liter of 10X TBE
electrophoresis buffer.
2. Stir to mix.
Recipes for Reagents and Stock Solutions
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Copyright © 2005, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.
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