Definitions and names
dH2O = building deionised water (from taps at each sink)
Milli-Q water = ultrapure water from the Lougheed Lab); don’t use unless specified as the filters are expensive!
Don’t confuse monobasic K phosphate (KH2PO4) with dibasic K phosphate (K2HPO4).
SDS (sodium dodecyl sulfate) = sodium lauryl sulfate.
Glycerol = glycerin
Tris = Tris-hydroxyaminomethane
How to pH a solution
All solutions with defined pHs should be checked using the pH meter. pH paper is not accurate enough! The pH should be accurate to within 0.1 pH units.
If you are not sure how much acid or base you will need, make up the solution in a volume smaller than the final volume (e.g. 800 ml for 1 liter), adjust the pH and then bring to the final volume.
To adjust pH, use concentrated HCl (strong acid, kept in the Acids cabinet) or 10 N NaOH (strong base). You can also use dilute HCl (1 M) or 1 N NaOH for fine adjustment of pH. Don’t use NaOH pellets unless specified.
Adjust pH while the solution is being stirred in a beaker. Add the acid or base in small increments and check pH after each increment so as not to overshoot.
Concentrated HCl is a 37% solution (~11 M). To make 1 M HCl, dilute 86.5 ml of conc. HCl in 1 liter dH2O.
1. Common lab solutions and media
Sterile ddH2O
Water from Milli-Q filter in Lougheed lab
Aliquot to 100 ml bottles and autoclave
Or Use the distilled Water from the Ko Lab and autoclave.
S Basal
(= "S Buffer")
To make 1 liter
NaCl 5.8 g [final] = 0.1 M
1 m K phosphate pH 6 50 ml [final] = 0.05 M
Cholesterol stock 1 ml
Make up in dH2O.
You will need:
1M K phosphate buffer, pH 6.0
To make 1 liter
KH2PO4 136.1 g
KOH 17.9 g
Dissolve in ~750 ml dH2O
Check pH and adjust if necessary (it should be close)
Bring volume to 1l with dH2O, aliquot to 500 ml bottle, autoclave
Alternatively, you can use:
POTASSIUM PHOSPHATE BUFFER (for NGM plates)
1) Make the following two solutions separately, then mix to obtain correct pH (6.0):
A. 250 ml 1M KH2PO4 (monobasic)Add dH2O to 34.0 g KH2PO4 until final volume (250 ml) is obtained
B. 200 ml 1M K2HPO4*H2O Add dH2O to 45.6 g K2HPO4 *Make sure the salt in the solutions is completely dissolved.
2) Add K2HPO4 solution to KH2PO4 solution to bring the pH up from 4.0 to 6.0 (will take about 100 ml of K2HPO4).
3) Divide phosphate buffer into aliquots of 75 ml.
4) Autoclave 15 min. liquid cycle 5) Store at room temperature.
Buffer Principle (link to flash movie)
Cholesterol stock solution
Make up a 10 mg/ml stock in 95% Ethanol; takes a while to dissolve; do not autoclave. Keep away from flame!
M9 (= "M9 salts" in Maniatis A.3)
To make up 1 liter:
Na2HPO4 5.8 g
KH2PO4 3.0 g
NaCl 0.5 g
NH4Cl 1.0 g
Make up in dH2O to 1 liter
Aliquot to 100 ml bottles
Autoclave (do not autoclave for longer than 30 minutes)
From the Koelle Lab:
M9 (without glucose or MgSO4):
MRC recipe; is basically M9 for bacteria without 20% glucose. used as M9 in Horvitz lab, e.g. for egg-laying assays, everything else. Not same as M9 buffer in worm book. (This is what we have been using)
Na2HPO4 5.8 g
KH2PO4 3.0 g
NaCl 0.5 g
NH4Cl 1.0 g
dH20 to 1l
M9 buffer (Worm book recipe):
also MRC recipe; Is basically M9 buffer with high NaCl to make up osmolarity for no glucose. Not used in Horvitz lab.
KH2PO4 3.0 g
Na2HPO4 6.0 g
NaCl 5.0 g
1M MgSO4 1.0 ml
dH20 to 1l
B broth
(Broth for E. coli OP50) /O:P>
To make up 1 liter
Bacto tryptone 10 g
NaCl 5 g
Add dH2O to 1 liter
Dissolve by stirring over heat
Aliquot to 100 ml bottles and autoclave.
LB Medium (for all other E. coli)
To make: 1 l 4 l
Bacto Tryptone 10 g 40 g
Bacto Yeast extract 5 g 20 g
NaCl 10 g 40 g
dH2O 800 ml 3200 ml
Adjust pH from ~6.9 to 7.5 with 12-14 ml of 1 M NaOH
Bring volume to 1l or 4 l
Aliquot 100-200 ml per bottle and autoclave.
2XTY
For 1 Litre
Tryptone 16g
Yeast Extract 10g
NaCl 5g
H2O to 1 litre
(2XTY Amp add 100ul (75mg/ml Amp) for every 100ml 2XTY)
Freezing Solution
(recipe from worm book, p.590)
To make up 1 liter
NaCl 5.85 g [0.1 M]
KH2PO4 6.8 g [0.05 M]
Glycerol 300 g 30%
1 m NaOH 5.6 ml
Weigh out the glycerol first in the flask.
Make up in dH2O to 1 liter aliquot into 100 ml and autoclave add MgSO4 once cool (see below)
Note: Mg++ must be added to a final concentration of 0.3 mM before use (add 30 ul of 1 M MgSO4 per 100 ml)
See also WormBook
2. Common solutions for molecular biology (Room Temperature)
2.1 Tris buffers
We keep the following lab stocks of Tris buffers: 1 M Tris pH 6.5, 7.0, 8.0, 8.9 and 2 M pH 7.5
1 M Tris (pH as desired)
To make 1 liter:
121.1 g Tris base
800 ml Milli-Q H2O
Dissolve Tris in H2O and adjust pH with conc. HCl:
? ml for pH 6.5
? ml for pH 7.0
~42 ml for pH 8.0
? for pH 8.9
Bring to 1 liter with Milli-Q H2O; don’t autoclave.
2 M Tris pH 7.5
To make 1 liter:
242.2 g Tris base
600 ml Milli-Q H2O
Dissolve Tris in H2O and adjust pH with ~ 125 ml conc. HCl
Bring volume to 1 liter with Milli-Q H2O; don’t autoclave
2.2 Solutions for minipreps and Qiagen preps
2.2.1 Stock solutions used to make Qiagen buffers:
Note: it is critical that these solutions are prepared accurately!
0.5 M MOPS pH 7.0
To make up 2 liters
209.3 g MOPS free acid
~750 ml dH2O
adjust pH to 7.0 with 10 N NaOH
make up to 2 liters with dH2O
Store at RT, do not autoclave
3 M NaCl
To make up 2 liters
350.6 g NaCl
Make up to 2 liters with dH2O
Store at RT
1 M Tris pH 8.5
To make up 1 liter
121.1 g Tris base
~750 ml H2O
Adjust pH to 8.5 with ~20 ml conc. HCl
Adjust volume to 1 liter
Store at RT
2.2.2 Buffers for use with Qiagen tips
QC For 1 liter
3 M NaCl 333 ml
0.5 M MOPS pH 7 100 ml
Isopropanol 150 ml
Add dH2O to 1 liter
Adjust pH to 7.0 with NaOH or HCl if necessary (it should be close)
QF For 1 liter
3 M NaCl 416.5 ml
1 M Tris pH 8.5 50 ml
Isopropanol 150 ml
Add dH2O to 1 liter
Adjust pH to 8.5 with NaOH or HCl if necessary (it should be close)
QBT For 1 liter
3 M NaCl 250 ml
0.5 M MOPS pH7 100 ml
Isopropanol 150 ml
Triton X-100 1.5 ml
Add dH2O to 1 liter
Solutions for Qiagen DNA preps
P1 solution 107 mL
1M Tris, pH 8.0 5 mL
0.5M EDTA, pH 8.0 2 mL
H2O 100 mL
Autoclave
Rnase A (add after autoclaving) 0.0107 g
Store at 4° C
If there is plenty of RNA in the preps then add RNase to 0.1mg/ml
P2 solution (lysis)
P2 = 200 mM NaOH, 1% SDS
P2 solution should be made fresh every 4 weeks.
To make up 50 ml from stock solutions:
1 ml 10 N NaOH
2.5 ml 20% SDS
36.5 ml dH2O
P3 solution (neutralising)
P3 = 3 M Potassium acetate pH 5.5
To make 1 liter
KOAc 294.5 g
Dissolve in 500 ml dH2O
Adjust pH to 5.5 with about 110 ml glacial acetic acid (kept in acid cabinet).
Adjust volume to 1 liter with dH2O. Keep at RT.
2.2.3 Other stock solutions for DNA minipreps
7.5 M Ammonium acetate
To make 1 liter:
578.1 g NH4Ac
Dissolve in dH2O to final volume of 1 liter.
Check the pH; it should be about 7.4
Store at RT
10% SDS
To make 1 liter:
100 g electrophoresis grade SDS (sodium dodecyl sulfate)
Add to 900 ml Milli-Q H2O
(To avoid spreading SDS dust, measure SDS into a closeable container in a fume hood--a 50-ml orange capped tube holds about 25 g-- and close the container before taking it to the balance.)
Place the container in a 65° water bath (with frequent stirring) to dissolve the SDS.
When SDS has dissolved, bring volume to 1 liter with Milli-Q H2O.
0.5 M EDTA pH 8.0
For 1 liter
186.1 g Na2EDTA.2H2O
800 ml Milli-Q H2O
Bring pH to 8 with NaOH pellets (about 20 g); the EDTA will not dissolve until the pH is about right. Bring volume to 1 liter with dH2O and autoclave.
10 N NaOH
400 g NaOH per liter dH2O
2.3 Buffers for gels
50X TAE
TO MAKE: 1l 2l 4l
Tris base 242 g 484 g 968 g
0.5 M EDTA pH 8.0 100 ml 200 ml 400 ml
glacial acetic acid 57.1ml 114.2 ml 228.4ml
dH2O to nearly... 1 l 2 l 4 l
Check the pH and if necessary adjust to 8.5 with glacial acetic acid
Bring to final volume with dH2O. Do not autoclave.
1X TAE running buffer is made up in the 20 liter carboy:
400 ml 50X TAE stock
dH2O to 20 liters
Add Ethidium bromide (EtBr) to the Gels NOT the running buffer.
Add 5ul (10mg/ml) EtBr per 100ml TAEl (caution: mutagenic)
10X TBE To make 4 liters
432 g Tris base
220 g Boric acid
160 ml 0.5 M EDTA pH 8.0
dH2O to 4 l
5x loading dye for agarose gels
50% glycerol 50 ml
0.5 M EDTA pH 8 10 ml
1 M Tris pH 7.5 5 ml
Add a tiny amount (a few mg) of the dye xylenol orange--just enough to color the solution
Add 35 ml dH2O to total volume 100 ml.
6X Loading Dye (Fermentas):
0.09% bromophenol blue (just add enough until you get a color you like)
0.09% xylene cyanol FF
60% glycerol
60mM EDTA
10X PBS
Phosphate buffered saline, recipe from Harlow and Lane.
To make up 1 liter
NaCl 80 g
KCl 2 g
Na2HPO4 14.4 g
KH2PO4 2.4 g
Dissolve in 800 ml dH2O
Adjust pH to 7.4 with HCl
Bring volume to 1 liter, autoclave
3. Worm plates
3.1 Standard worm (NGM agar) plates
To make: 1 liter 3 liters
NaCl 3 g 9 g
Bacto-agar 17 g 51 g
Bacto-peptone 2.5 g 7.5 g
Cholesterol 1 ml 3 ml
dH2O ~975 ml ~2925 ml
Make up 3 liters in a 6 liter Erlenmeyer flask. Also autoclave 500ml of dH20 to rinse out tube and pump after pouring.
Autoclave 60 min. (#3 program 2nd Floor Biosciences) on liquid cycle (depending on how many liters are being autoclaved), then using sterile technique add the following:
1 m CaCl2 1 ml 3 ml
1 m MgSO4 1 ml 3 ml
1 m KPO4 pH 6 25 ml 75 ml
Swirl to mix thoroughly after each addition; after all additions done, pour plates.
To make up the stock solutions (Keep Solutions Sterile):bbsp;
1 m CaCl2
To make 1 liter
110.9 g CaCl2
dH2O to 1 liter
Aliquot 100 ml/bottle, autoclave
1 m MgSO4
To make 1 liter
120.3 g MgSO4
dH2O to 1 liter
Aliquot 100 ml/bottle, autoclave
Aliquot to 250 ml bottles, autoclave on liquid cycle
3.2 Agarose plates
(for worm genomic DNA preps)
For 4 litres
NaCl 12 g
Agarose 60 g
Bacto Peptone 30 g
Make up in 4 l dH2O. Autoclave for 45’, allow to cool to about 55°C, then add:
0.5 M CaCl2 8 ml
1 m MgSO4 4 ml
1 m K phosphate pH 6 100 ml
cholesterol 4 ml
Let dry for one-two days at RT.
4. Bacterial plates
All plates must be made with strict sterile technique. After pouring, leave plates to dry at RT for 1-2 days, then store upside-down in plastic bags in the cold room. Plates should be clearly marked on the outside with colors indicated. Close the bags with tape, mark and date each bag.
LB plates
To make 4 litres:
40 g Bacto Tryptone
20 g Bacto Yeast extract
40 g NaCl
Make up in 3200 ml dH2O.
Adjust pH from ~6.9 to 7.5 with 12-14 ml of 1 M NaOH
Bring volume to 4 l.
Add 60 g Bacto-agar, mix, autoclave.
When agar has cooled to 55°C, pour plates (~25 ml per large plate). If you can keep your hand on the side of the flask then it is cool enough.
Mark LB sides with GREEN stripe.
LB Amp plates (75 mg/ml Ampicillin)
Follow protocol for LB plates until after autoclaving. When agar has cooled to 55°C, add 4 ml (2 vials) of 75 mg/ml Ampicillin stock (1000X stock, kept at -20°C). Pour plates as above. Amp plates marked with RED stripe.
Ampicillin is unstable. Plates older than four months cannot be used--so don’t make up too much at any one time.
LB Kan plates (50 mg/ml Kanamycin)
As for LB Amp plates except add 4 ml of 50 mg/ml kanamycin stock (1000X stock, kept at -20°C).
M9 Minimal plates
To make up 1 litre
15 g Bacto-agar
750 ml dH2O
Autoclave and let cool to 50°C
Add using sterile technique:
5x M9 salts 200 ml
1 M MgSO4 2 ml
20% glucose 20 ml
1 M CaCl2 0.1 ml
Swirl to mix well; pour plates, leave to dry 2 days at RT then store in cold room.
To make up additions:
5x M9 salts
To make up 1 liter
Na2HPO4.7H2O 64g
KH2PO4 15g
NaCl 2.5g
NH4Cl 5 g
Dissolve in 1 liter dH2O, divide into 5 x 200 ml aliquots and autoclave for 15 mins.
Make up 20% glucose in dH2O and filter-sterilise.
5. Yeast media
YPAD plates
To make 1 liter
10 g yeast extract
20 g peptone
48 mg Adenine hemisulfate (For YPD plates leave this out)
20 g dextrose
20 g agar
dH2O to 1 liter
Autoclave
Synthetic media plates (-LEU, -TRP, etc.)
To make 1 liter
10 g yeast nitrogen base (without amino acids with ammonium sulfate)
20 g dextrose
20 g agar
dH2O to 1 liter
Autoclave for 40 minutes and take out as soon as cycle over; immediately add the appropriate synthetic powder (amount on side of bottle) and mix.
Note you can make drop out mixes from existing synthetic media, for example you can make -LEU, -TRP media by adding HIS to a -LEU -TRP -HIS triple drop out synthetic media.
For Yeast 2 Hybrid Screening (not for general use)
3-AT (3-amino, 1,2,4-triazole) addition:
Let agar cool to 50° then add 3-AT powder to media.
For 25 mM 3-AT, add 2.1 g/l
For 50 mM 3-AT, add 4.2 g/l
6. Molecular Biology stock solutions (kept at -20°C)
Salmon sperm DNA 10 mg/ml
500 mg salmon sperm DNA
50 ml Milli-Q dH2O
dissolve by heating or stir overnight at 4C.
(Does not have to be sheared by sonication-works fine without)
Store at -20°C. BOIL for 10 minutes (once is fine) before using.
50 x Denhardt's
For 500 ml
5 g Ficoll
5 g polyvinylpyrrolidone
5 g BSA (Pentax Fraction V)
500 ml Milli-Q H2O.
Filter sterilize, 10 ml aliquots, store at -20°C.
1 M DTT
For 60 ml
9.27 g dithiothreitol
0.2 ml 3 M NaAc pH 5.2
59.8 ml dH2O
Filter sterilise, store 1 ml aliquots at -20°C.
2% ITPG (~84mM)
2% IPTG (isopropyl b-D-thiogalactopyranoside):
20 mg/ml IPTG in double distilled water
200 mg IPTG (Sigma I-5502)
ddH2O to 10 ml (aliquot and store at -20degC)
For 1M stock add 2.38g per 10ml H2O. Induce cells with concentrations raging from 0.1mM to 1mM IPTG.2% X-Gal (in DMSO or DMF) (~49mM)
X-gal (5-bromo-4-chloro-3-indolyl b-D-galactopyranoside):
200 mg x-gal (Sigma B-4252)
dimethylformamide (DMF) to 10 ml
Aliquot and store protected from light at -20degC)
For LB AMP plates we want about 40ug/ml X-gal. So spread 50ul (X-gal) to each plate. (Each plate has about 25ml) 200 mg x-gal (Sigma B-4252)
Antibiotic stock solutions (75mg/ml Amp, 50 mg/ml Kan)
Make up in Milli-Q H2O (50%) and 50% EtoH. This avoids multiple freeze thaw cycles because the Amp will not freeze. Do not autoclave! Filter-sterilise (not necessary) store 1 ml aliquots at -20°
Tetracycline stock (Tet): Stock of 10 mg/ml in 50% ethanol + sddwater.
1 g Tetracycline (Sigma T-3383)
50 ml 100% ethanol
ddH2O to 100 ml (store at -20C)
7. Other
Diatomaceous earth (100 mg/ml):
Suspend 10 g of diatomaceous earth (Sigma D-5384) in 100 ml of distilled water in a 100 ml graduated cylinder, and let it settle down for 3 hours. Decant the supernatant, and resuspend the pellet in 100 ml of 6 M guanidine HCl, pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA.
Diatomaceous earth-wash buffer:
10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 50% ethanol in double distilled water.
10 ml 1 M Tris-HCl, pH 8.0
2 ml 0.5 M EDTA, pH 8.0
500 ml 100% ethanol (McCormick Distilling Co., Inc.)
dH2O to 1 L
DNase-free RNase A:
10 or 20 mg/ml RNase A in 1 mM NaOAc, pH 4.5.
100 or 200 mg RNase A (Sigma R-5500)
3.3 ul 3 M NaOAc, pH 4.5
dH2O to 10 ml
boil for 10 minutes (aliquot and store at -20deg.C).
10 mg/ml ethidium bromide (EtBr):
1 g EtBr (Sigma E-8751)
ddH2O to 100 ml
Try making a smaller volume if possible
1 M HEPES, pH 7.5:
23.83 g HEPES (Sigma H-3375)
ddH2O to 100 ml
adjust pH to 7.5 with potassium hydroxide (KOH) (store at 4deg.C).