Reagents Used in Molecular Lab

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°

0.5 N NaOH
1.5 M NaCl

*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°

2X SSC

*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

70% v/v with water

*gelatin, 2% - store at RT°

2g gelatin
100 ml water
autoclave

*glacial acetic acid - store at RT°

100% acetic acid

*glucose, 10% - store at RT°

70 ml water
10 g glucose
water to 100 ml
sterilize by filtration

*glycerol, sterile RT°

autoclave

*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°