Wormlab Recipe Book

Wormlab recipe book

(Modified from Jin and Chisholm Labs)

Revised 07/13



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 H2

            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 H2

            (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;          

m CaCl2

            To make 1 liter 

            110.9 g CaCl2

            dH2O to 1 liter

            Aliquot 100 ml/bottle, autoclave 

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).

 

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