Acrylamide stock (38%/2%) (Hazardous to your health. Wear gloves. Work in hood.)
76 g acrylamide
4 g methylene bisacrylamide
Make up to 200 ml with DDH2O. Store in dark at 4¡. (Wrap bottle with tin foil.)
AQUA Hyb.(500ml)
125ml 20 X SSC (5X SSC)
50ml 50X Denhardts (5 X Denhardts)
10ml 0.5M EDTA (10mM EDTA)
315ml DDW
Ampicillin
Stock = 100 mg/ml
1 g ampicillin sodium (pharmaceutical grade) in 10 ml sterile DDH2O
Store in aliquots at -20¡
Denhardt's Reagent (50x stock)
Ficoll 5g
Polyvinyl pyrolidone 5g
Bovine Serum albumin 5g
DDW to 500mls
Dissolve, aliquot 40ml into 50ml tubes and freeze at -20¡C
Dialysis tubing (4 meters tubing)
Use Spectrapor (MW c/o) 6-8000
1. Simmer tubing for 1 hr in 2 Lts 50% ethanol. Pour off and simmer tubing for 1hr in 2 Lts 10 mM NaHCO3 (1.68g), 1mM EDTA (4ml 0.5M EDTA). Change buffer after 30mins
2. Simmer tubing for 1hr in 2 Lts DDW. Change DDW after 30 mins
3. Store tubing in 10% ethanol 0.5mM EDTA at 4¡C (0.5ml 0.5M EDTA/500ml)
Touch membrane with gloved hands only. Do not let membrane dry out once wet. Before use wash in DDW and flush through with DDW. Check membrane for leaks before loading sample.
2x Freezing Medium (1lt)
NaCl 3g
K2HPO4 (anhyd) 12.6g
Na citrate. 2H2O 0.9g
MgSO4. 7H2O or Mg SO4 0.18g or 0.09g
(NH4)2 SO4 1.8g
KH2PO4 (anhyd) 3.6g
Glycerol 70ml@88g
DDW to 1 Lt
Autoclave
Store 4o
Kan Plates
5 g NaCl
10 g Bactotryptone
5 g yeast extract
15 g agar
DD H2O to 1 liter
Add 1 ml Kanamycin stock before pouring.
Kanamycin Stock
Stock: 5 g Kanamycin (Sigma) in 100 ml H2O
Filter sterilize
add 1 ml / liter plates
Lambda Plates (1lt )
NaCl 5gms
Casein Hydrolysate 10gms
Yeast Extract 5gms
Agar 15gms
DDW to 1lt
400ul 5M NaOH
Stir
Autoclave
Pours 20 large plates.
L-amp Plates: (1lt )
NaCl 5gms
Casein Hydrolysate 10gms
Yeast Extract 5gms
Agar 15gms
Autoclave
75mg/L ampilillin when agarose has cooled to about 45¡C.
LMM: (1 lt)
NaCl 5g
Casein hydrolysate 10g
Yeast extract 5g
Maltose 2g
MgCl2 2g
DDW to 1lt
Autoclave
LBroth (1lt)
Casein hydrolysate 10g
Yeast extract 5g
NaCl 5g
DDW to 1lt
Autoclave
Markers
Hind III Markers
50ul l DNA (1mg/ml) )Pharmacia 500ug 27-4111-01
20ul 10 x buffer
2.5ul HIND III
127.5ul dH2O
digest O/N at 37¡C
Heat inactivate at 70¡C for 10 minutes
add 40ul loading dye
pUC digested with HPA II
As per Hind III Markers but after digest phenol / chloroform extraction with 200ul, vortex, spin 2 minutes. Transfer upper layer to eppendorf tube
Heat inactivate at 70¡C for 10 minutes
Add: 400ul dH2O
50ul loading dye
37.5ul PVC / HPA II
Minimal Plates
for 500 ml: Autoclave 7.5g agar in 419 ml DDH2O with stir bar.
Add following sterile solutions:
50 ml 10xM9 phosphate
5 ml 10% NH4Cl
5.5 ul 0.1 M MgSO4
20 ml 10% glucose
1 ml 10 mg/ml thiamine HCl (add when ready to pour)
10X M9 Phosphate
30 g KH2PO4
113g Na2HPO4 ¥ 7 H2O / to 1 liter H2O
Autoclave
250 mM EDTA pH 8.0
186.1 g Na2 EDTA ¥ 2 H2O
Dissolve in ~1700 ml DD H2O. Adjust pH with 50% NaOH to pH 8.0 (~28 ml 50% NaOH). EDTA won't dissolve until the pH is increased. Make up to 2 liters with DD H2O. Autoclave.
10x MOPS
MOPS 20.93g
0.5M EDTA 10ml
3M Na accetate 8.33ml. Adjust pH to pH7, use ~2g NaOH
Nucleotide triphosphates
To 25 mg nucleotide triphosphate add 350ul sterile water. Bring to pH 7 by adding 1N NaOH (20-50 ul), using pH paper to test after each addition. Make a 1/2000 dilution into a buffer of the appropriate pH and measure the optical density at the appropriate wavelength. The final concentration should be about 100mM.
Nucleotide pH of Wavelength Extinction
dilution buffer coefficient
dATP 7 259 nm 15.2 x103
ATP 7 259 15.4 x103
dCTP 2 (0.01 M HCl) 280 13.1 x 103
CTP 2 280 12.8 x 103
dGTP 7 253 13.7 x 103
GTP 7 253 13.7 x 103
dTTP 7 267 9.6 x 103
UTP 7 262 10.0 x103
NOTE: conc. = optical density/extinction coeff. x dilution factor. So for a 100 mM solution of UTP, diluted 1/2000 in pH 7 buffer, the optical density at 262 nm will be 0.5: 10-1 M = (0.5 O.D. units/10x103 M/O.D.) x (2x103).
Phenol
1. Dissolve a bottle of crystalized phenol in a water bath 68¡C. Measure vol. when dissolved.
2. To the melted phenol add 0.1% hydroxyquinaline i.e. 1 gm per 1000 mls
3. To adjust the pH add equal vol. of 0.5M Tris. Cl pH8.
4. Stir with flea 15' then allow phases to separate. Aspirate top layer in hood. Add equal vol. 0.1M Tris. Cl pH8.
5. Stir 15', stand to separate phases then aspirate. Repeat extractions until pH of phenolic phase is > 7.8. Add 0.1 vol. of 0.1M TrisCl. Add equal vol. chloroform.
PBS (Phosphate buffered Saline) (20lt)
NaCl 180g
Na2HPO4 113.57g
KH2PO4 27.20g
Basic PLC lysis buffer (500 ml)
50 mM Hepes pH7.5 25ml 1M Hepes pH 7.5
150 mM NaCl 4.38g
10% glycerol 60g
1% Triton X100 5g
1 mM EGTA 2ml 0.25M EGTA
1.5mM MgCl2 0.15g or 0.75ml of 1M MgCk
Additives:
Protease inhibitors
1. Aprotinin (Boehringer, #981532) 10ug/ml. 10mg/ml stock in PBS as aliquots at -20¡C (avoid repeated freeze thawing).
2. Leupeptin (Boehringer, #1017 101) 10ug/ml 10mg/ml stock in DDW as aliquots at -20¡C (avoid repeated freeze thawing).
3. Pepstatin (Boehringer, #253 286) 0 5ug/ml. Prepare as a 1mg/ml stock in methanol
4. PMSF 1mM (see below for preparation of stock)
Phosphatase inhibitors
1. b-glycerophosphate 50mM
2. Sodium Orthovanadate 0.1M (see below for preparation of stock).
3. NaFluoride 100mM
PMSF:
To prepare PMSF dissolve at 0.1M in ethanol (17mg/ml ethanol = 0.1M). Add very slowly to stirring PLC buffer immediately before added to cells. Boerhinger 837091
Na Orthovanadate, 100mM solution:
1. 0.92g (Sigma S-6508) Na3VO4
40ml DDW. pH will be about 12 when dissolved. Adjust with HCl to 10.
2. Place in boiling water bath for 5' and cool on ice. Repeat boiling and cooling till color has gone. The pH will be much lower now. Adjust to 10 with NaOH and repeat boiling and cooling until yellow color has gone and pH is stabilized at 10.
3. Make to a final volume of 50ml with DDW and aliquot and freeze at -20¡C.
Wally Langdons lysis buffer
Wash buffer for cells
10mM Tris pH 7.4
150mM NaCl
Lysis buffer
20mM Tris pH 8.0
150mM NaCl
1mM EDTA
1% TX100
Protease and phosphatase inhibitors as above.
RNASE A
Dissolve pancreatic RNASE A at 10mg/ml in 10mM Tris, HC1 (pH 7.5),15mM NaCl.
Heat to 100¡C for 15'
Allow to cool slowly to room temperature.
Aliquot and store at -20oc.
2.5 x TBE Sequencing buffer
230gms urea
62.5mls 20xTBE
75mls 40% acrylamide, no urea
25gms sucrose
25mg = 0.025gm Bromophenol Blue
DDW to 500mls
0.5 x TBE sequencing buffer
460gms urea
25mls 20 x TBE
140mls 40% acrylamide, no urea
DDW to 1 Litre
20X SSC
350.6 g NaCl
176.5 g Sodium Citrate
Dissolve in 2 liters DD H2O.
Autoclave.
20X SSCP
800 ml 1M NaH2PO4 pH 6.8
176.4g Trisodium Citrate
280.5g NaCl
to 2 liters with DD H2O.
Autoclave.
10 x SOC broth for electroporation
per 100 ml per 500 ml
MgCl2 6H2O 2.0g 10g
MgSO4 7H2O 4.9g 24.5g
KCl 180 mg 900 mg
glucose 3.6g 18g
Prepare in dH2O. Filter sterilize, do not autoclave.
Dilute to 1 X in LMM.
Southern Blot Sol'ns
4 liters
Denat. 0.5M NaOH 200 ml 10N NaOH
1.5M NaCl 350.6 g NaCl
Neut. 0.5M Tris pH 7.4 242.2 g Tris pH 7.4
1.5M NaCl 350.6 g NaCl
pH with >250 ml conc HCl
TAE (Tris Acetate EDTA). 1 x = 0.04M Tris Acetate, 0.001M EDTA
50X stock of 2lt
242g Tris base
57.1 mls glacial acetic acid
100mls 0.5M EDTA pH8
20X TBE
432 g Tris base
220 g Boric acid (crystal dissolves easier than powder)
40.8 g Na2 EDTA ¥ 2 H2O
Dissolve in 2 liters DD H2O
Check: pH = 8.35 for a 1:20 dilution
Autoclave. (Use Bellco 1 liter bottles)
TBS 1LT
137mM NaCl 8g/Lt
3mM KCl 0.2g/Lt
25mM Tris pH8 25ml 1M
TE
10mM Tris pH 7.5 + 1mM EDTA
100ml
IM Tris 1ml
500mM EDTA 0.2ml
Tetracycline
5 g tetracycline (tetracycline HCl, pharmaceutical grade)
100 ml 50% Ethanol / 50% sterile DDH2O
Want 10 ug/ml for liquid media, 12.5ug/ml for solid media
Add 0.25 ml/500 ml L-broth
Let agar stir a while after adding antibiotic.
NOTE. Tetracycline is light sensitive. Store in dark. Media made with tetracycline should lack magnesium as this interfers with tetracycline's action.
TM10 with Gelatin
2.41 g MgSO4
20 ml 1 M Tris pH 7.5
20 ml 1% gelatin (or 0.2g dry gelatin. Heat to dissolve.)
DD H2O to 2 liters.
Autoclave.
TM10 - 10 mM MgSO4 and 10 mM Tris pH 7.5
2.41 g MgSO4
20 ml 1 M Tris pH 7.5
Make up to 2 liters with DD H2O. Autoclave.
Terrific broth / K2K (1lt)
Casein hydrolysate 12g
Yeast extract 24g
Glycerol 5g
DDW to 900 ml. Autoclave
K2K for terrific broth
KH2PO4 2.31g
K2HPO4 12.54g (16.47g K2HPO4.3H2O)
DDW to 100 ml
Autoclave. Add to above broth when cool
Top Agarose:
NaCl 3gms
Casein Hydrolysate 6gms
Yeast Extract 3gms
Mg SO4 0.72
DDW 600mls
Agarose 1.4 x 3 * Add agarose individually to each bottle then AUTOCLAVE.
Quick Top Agarose:
100mls of LMM broth
add 0.7gms of agarose
Dissolve agarose and wait till temp 45¡C. It can then be poured onto plates. Large Plates 12mls
Small Plates 4mls
Top Agarose with Extras (IPTG/ XGal and Amp):
For 10mls of top agarose add:
100ul X gal (20 mg/ml)
100ul IPTG (20 mg/ml)
15ul 100mg/ml AMP
2 x YT (1lt)
Casein hydrolysate 16g
Yeast extract 10g
NaCl 10g
DDW to 1lt . Autoclave