APPENDIX I
List of Reagents, in alphabetical
order
(* reagents used in Mol. Bio. Lab)
*Reagent - Storage Condition
*2X YT - store at room temp.
- 900 ml water
- 16 g Bacto tryptone
- 10 g Bacto yeast extract
- 5 g NaCl
- Shake until dissolved and pH with 200 µl
5 M NaOH
- adjust volume to 1 L
- autoclave
*agar plates
- store at room temp.
- to 1 L of any medium, add 15 g of Bacto Agar
- autoclave to sterilize, pour while hot, rinse
flask well before it hardens
*ammonium acetate, 7.5 M - store at room temp.
- 50 ml water
- 57.75 g ammonium acetate
- adjust volume to 100 ml with H2O
*ampicillin (Fisher
#BP1760-25) - store at -20° C
- 100 mg/ ml water - this is a 1000X stock solution
- filter sterilize with a 0.22 µm filter
into a sterile container
*Blotto- store at 4° C
- 7 g Dry Milk
- 10 mL 10X Tris Saline Stock
- 100 µL Tween 20
- 100µL Antifoam A
- Water to 100 mL
*CaCl2 solution - store at
+4° C
- 60 mM CaCl2
- 15% Glycerol
- 10 mM PIPES, pH 7
- Autoclave.
*chloroform, 100% - store at RT°
- already prepared
WARNING THIS IS A KNOWN MUTAGEN WEAR GLOVES
*competent bacteria (capable of taking up DNA)
- store at -70° C
- already prepared (CaCl2 method)
*denaturing solution (for DNA) - store at RT°
*DNA loading dye - store at +4° C
- for 10 ml of a 10X stock solution:
- 30% glycerol (v/v)
- a pinch of bromophenol blue
- a pinch of xylene cyanol FF
*DNAse, 10 mg/ ml - store at -20° C
- CAUTION - THIS WILL DEGRADE ALL DNA SAMPLES SO
BE CAREFUL
- dilute the bottle of DNAse with sterile water
so that the final concentration is 10 mg/ml (e.g. 10 mg plus 1 ml of water).
- Once it is dissolved, aliquot into 100 µl
volumes and store at -20° C
*ECL Primary Wash- store at RT°
- 0.4 % SDS (start with 20% SDS stock solution)
- 0.5X SSC
*ECL Secondary Wash- store at RT°
*EDTA, 0.5 M pH 8.0 - store at RT°
- 93.0 g EDTA
- 350 ml of water and stir on magnetic stirring
plate
- about 10 g NaOH pellets
- add 5 M NaOH until all EDTA is dissolved (this
happens at pH 8.0)
- adjust volume up to 500 ml with water
*ethidium bromide - store at +4° C
WARNING THIS IS A KNOWN MUTAGEN WEAR GLOVES
- already prepared
- 10 mg/ ml in H20
*EtOH (ethanol) - store at RT° or +4° C
- unless stated otherwise, it is 100% and from
a "DNA only" bottle
*EtOH 70%, - store at RT°
or +4° C
*gelatin, 2% - store at RT°
- 2g gelatin
- 100 ml water
- autoclave
*glacial acetic acid - store at RT°
*glucose, 10% - store at RT°
- 70 ml water
- 10 g glucose
- water to 100 ml
- sterilize by filtration
*glycerol, sterile RT°
*IPTG (isopropylthio- b - D -galactoside) -
store at -20° C
- 8 ml water
- 2 g IPTG
- adjust volume to 10 ml
- sterilize by filtration, aliquot in 1 ml volumes
and store at -20° C
*LB - store at RT°
- for 1 L of LB medium:
- 700 ml water
- 10 g Bacto Tryptone
- 5 g Yeast Extract
- 10 g NaCl (5 g for low salt version)
- 200 µl 5 M NaOH
- water up to 1L
- Autoclave in 2 X 1 L bottles, 500 ml in each
bottle
*magnesium sulfate, 1 M - store at RT°
- you should figure this one out
- MgSO4*7H20
FW = 246.48
- make 100 ml total volume
- autoclave
*maltose, 20% (w/v) - store at RT°
- 20 g maltose
- water up to 100 ml
- Sterilize by filtering through a 0.22 µm
filter into a sterile bottle
*miniprep solution I - store at RT°
- 15 ml of water
- 0.23 g glucose
- 0.626 ml 1 M Tris, pH 8.0
- 0.5 ml 0.5 M EDTA
- water up to 25 ml
*miniprep solution II - store at RT°
- 0.4 ml 5 M NaOH
- 9.1 ml water
- 0.5 ml 20% SDS
*miniprep solution III - store at +4° C
- 60 ml 5 M potassium acetate stock solution
- 11.5 ml glacial acetic acid
- 28.5 ml water
*neutralizing solution (for DNA) - store at
RT°
- 1.5 M NaCl
- 0.5 M Tris, pH 7.5
*Ponsceau S- store at RT°
- 0.1% w/v Ponsceau S
- in 1% v/v acetic acid
*phenol/chloroform/isoamyl alcohol (Ameresco) - store
at +4° C
- already prepared
- chloroform:isoamyl alcohol 24:1
- the above:phenol 1:1
- the phenol/chloroform is the bottom phase and
the buffer is on top
WARNING THIS IS A KNOWN MUTAGEN WEAR GLOVES
*phenol, saturated and buffered (pH 8.0) (Ameresco)
- store at +4° C
- already prepared but the phenol is the bottom
phase and the buffer is on top
WARNING THIS IS A KNOWN MUTAGEN WEAR GLOVES
*potassium acetate, 5 M stock - store at RT°
- 49.07 g potassium acetate
- water up to 100 ml
*proteinase K, 10 mg/ ml - store at -20° C
- CAUTION - THIS WILL DEGRADE ALL PROTEIN SAMPLES
SO BE CAREFUL
- dilute the bottle of proteinase K with sterile
water
- so that the final concentration is 10 mg/ml (e.g.
10 mg plus 1 ml of water).
- Once it is dissolved, aliquot into 100 µl
volumes and store at -20
*RNAse, 10 mg/ ml (SIGMA# R-5125) - store at
-20° C
- CAUTION - THIS WILL DEGRADE ALL RNA SAMPLES SO
BE CAREFUL
- dilute the bottle of RNAse with sterile water
so that the final
- concentration is 10 mg/ml (e.g. 10 mg plus 1
ml of water). Once it is dissolved,
- aliquot into 100 µl volumes and store at
-20° C
*sarkosyl (Sigma # L-5125) - store at RT°
- 20% w/v YOU FIGURE OUT AMOUNTS
- mix powder and water and stir for a long time
at RT°
*SDS (Sigma # L-5750) sodium dodecyl sulfate,
also called lauryl sulfate - store RT°
- 20% w/v YOU FIGURE OUT AMOUNTS
- mix powder and water and stir for a long time
at RT°
WARNING: DO NOT BREATHE ANY SDS DUST - IT WILL MAKE
YOU COUGH A LOT
*SM - store at RT°
- for 1 L of SM:
- 5.8 g NaCl
- 2.0 g MgSO4
.7H 2
O
- 50 ml 1 M Tris, pH 7.5
- 5 ml 2% gelatin
- water up to 1 L
- Sterilize by autoclaving in 2 X 500 ml bottles
*sodium acetate, 3M - store at RT°
- 40.8 g sodium acetate.3H20
- 50 ml water
- pH 5.2 use glacial acetic acid
- pH 7.0 use dilute acetic acid
*sodium chloride, 5 M - store at RT°
- you figure this one out
- FW = 58.44
- make 500 ml of this in water
*sodium hydroxide (CAUTION
- VERY CAUSTIC) - store at RT°
- you figure this one out
- FW = 40.0
- make 100 ml of this in water
*SSC, 20X stock - store at RT°
- 800 ml water
- 175 g NaCl
- 88 g sodium citrate
- adjust pH to 7.0 with conc. HCl
- adjust volume to 1 L
- autoclave
*TBE - Tris, Boric acid, EDTA - store at RT°
- for 1L of a "5X" stock:
- 54 g Tris base
- 27.5 g boric acid
- 20 ml 0.5 M EDTA
- H 2 O up to 1 L
*TE - Tris, pH 8.0, EDTA - store at RT°
- to make 50 ml of a 10X stock solution:
- 5 ml of 1 M Tris, pH 8.0
- 1 ml of 0.5 M EDTA
- 44 ml of H20
*TEN - store at +4°
C
- 150 mM NaCl
- 10 mM EDTA
- 10 mM Tris-HCl, pH 8.0
*Tetracycline - store at -20° C
- 5 mg/mL 100 ethanol (this is sterile due to ethanol)
- final concentration = 50 µg/mL for high copy plasmids (e.g. pSB1A3); stock is 100X
- 10 µg/mL for low or medium copy plasmids; stock is 500X
*top agarose or agar - store at RT°
- 0.7 g agarose
- 100 ml H20
- autoclave in 2 X 100 ml bottles
*10X Transfer buffer stock (western blots)- store
at RT°
- 30.3 g Tris Base
- 144.1 g glycine
- water to 1 L
*1X Transfer buffer (western blots)- store at 4°
C
- 100 mL 10X stock
- 500 mL water
- 200 mL methanol
- water to 1 L
- chill to 4° C
*Tris, 1 M - store at RT°
- 121 g Tris base
- 800 ml H20
- pH with HCl
- pH 7.5 takes ~ 60 ml HCl
- pH 8.0 takes ~ 40 ml HCl
- use pH meter to home in on correct pH
- H20 up to 1 L
- autoclave
*10X Tris Saline- store at RT°
- 6.05 g Tris Base
- 43.9 g NaCl
- water to 500 mL
*Turbo Denaturing Buffer- store at RT°
- 3 M NaCl
- 0.4 M NaOH
- water to 1 L
*Turbo Transfer Buffer- store at RT°
- 3M NaCl
- 8 mM NaOH
- 2 mM Sarkosyl
*5X Turbo Neutralizing Buffer- store at RT°
- 1 M phosphate buffer, pH 6.8
- 79.5 g Na2HPO4
- 52.39 g anhydrous NaH2PO4 or 60.25 g NaH2PO4.H2O
*X-gal (5-bromo-4-chloro-3-indoyl- b - D -galactoside)
- store at -20° C
- 20 mg/ml in dimethylformamide in a glass container
- wrap in foil
- store at -20° C
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© Copyright 2002 Department of Biology, Davidson College, Davidson, NC 28036
Send comments, questions, and suggestions to: macampbell@davidson.edu