SEALFON LAB BUFFER RECIPES
ROOM TEMPERATURE BUFFERS
0.1% DEPC treated water
Add 1 ml of DEPC to 1 liter of ddH2O water
Shake vigorously to bring the DEPC into the water.
Let the water stand for 12 hours at room temperature in the dark
Autoclaved for 20 minutes
4% formaldehyde
10mL Formaldehyde methanol-free from Polysciences (CAS: 50-00-0)
30mL H2O
sodium carbonate solution
10g NaCO3 (CAS: 497-19-8)
500mL H2O
Sodium potassium tartrate
10g NaK tartrate (CAS: 638-59-5)
500mL H2O
Copper sulfate 1%
5g CuSO4 (CAS: 7758-99-8)
500mL H2O
500mM Ammonium chloride
0.078g ammonium chloride (CAS: 12125-02-9)
30mL H2O
7.5M ammonium acetate
57.81g ammonium acetate (CAS631-61-8)
Up to 100mL H2O
2M sodium acetate
27.22g sodium acetate (CAS: 127-09-3)
Up to 100ml H2O
50X TEA
242g Tris base (CAS: 77-86-1)
136g sodium acetate (CAS 127-09-3)
18.6g Na2EDTA (CAS 139-33-33)
up to 1l H2O
0.5M EDTA (ethylenediamine-tetracacetic acid)
186.1g Na2EDTA (CAS: 139-33-33)
~50ml 1N NaOH solution
H2O up to 1l
1N NaOH
20g NaOH (CAS: 1310-73-2)
Up to 500ml H2O
70% Ethanol
35 ml 100% ethanol (CAS: 64-17-5)
15ml H2O
10XTBE (1 liter)
Tris base: 108g
Boric Acid: 55g
0.5M EDTA (pH 8.0): 40ml
Add ddH2O to make 1 liter
400mM Ammonium Sulfate (1 liter)
Ammonium Sulfate: 52.86g
Add ddH2O to make 1 liter
20XSSC (1 liter)
NaCl: 175.3g
Sodium Citrate: 88.2g
Add ddH2O to make 1 liter
20XPBS (1 liter)
NaCl: 160 g
KCl: 4 g
Na2HPO4: 28.8 g
KH2PO4: 4.8 g
Add ddH2O and adjusted the pH to 7.4, autoclaved.
Working solution of PBS was prepared by diluting the 20X stock solution to 1X with autoclaved ddH2O or DEPC treated water.
PMSM (1 liter)
1XPBS with 50 mM MgCl2: Dissolve 10.165g MgCl2 to 1XPBS.
1M MgCl2 (100ml)
MgCl2.6H2O: 20.33g
Dissolve in ddH2O to make 100ml solution
10%SDS
Sodium dodecyl sulfate: 10 g
Add ddH2O to 100ml
1M Trehalose (100ml)
Trehalose: 24.23g
Add ddH2O to make 100 ml
2M NAOAce (100ml)
Sodium Acetate.3 H2O: 27.2g
Dissolve in 80 ml ddH2O, adjust pH to 4.0 with glacial acetic acid, adjust volume to 100ml
10% Sarcosyl (100ml)
Dissolve 10g of sarcosyl-L-serine in ddH2O, adjust volume to 100ml
5M NaCl (100ml)
NaCl: 29.22g
Add ddH2O and adjust volume to 100 ml
1M Tris (1 liter)
Dissolve 121.1 g Tris base in 800 ddH2O, adjust pH by adding HCl
pH 7.4: 70ml HCl
pH 7.6: 60ml HCl
pH 8.0: 42ml HCl
Make up the volume to 1 liter, sterilize by autoclaving
50mM Glycine in 1XSSC (100ml)
Dissolve 0.375 g glycine in 1XSSC
0.1% Triton X-100 in PBS:
Add 100 ml Triton X-100 to 100 ml PBS
Stir gently with magnetic stirrer for at least 3 hours before use
AcNH4
Ammonium nitrate 7.7 M
DEPC water
NaCl 3M
175.29g/l
Store at RT
Stock binding buffer (x10):
200 mM HEPES (Sigma# H-3375)
1M NaCl (Fisher# S271)
30 mM MgCl2 hexahydrate (Sigma# M2670)
For 1000 ml:
47.6 g HEPES
58.4 g NaCl
6.1 g MgCl2
pH 7.4; store at 4C
Binding buffer (200ml)
Stock binding buffer 20ml
Ascorbic acid (20mM) 40_l (0.2mM final)
H2O 180ml
Supplement with proteinase inhibitor (Roche #1 873 580)
Check pH
Solubilization buffer (500 ml)
Final conc.
Tris-HCl 0.5M, pH 7.4 100 ml 100mM
NaCl 3M 33.3 ml 200mM
EDTA 1M 0.5 ml 1mM
Igepal CA-630 (*) 6.25 ml 1.25%
H2O Up to 500 ml
Supplement with proteinase inhibitor (5 pills; Roche #1 873 580)
Check pH
Depending on step
SDS (0.2%) 0.2 g/100 ml 0.2%
(*) Sigma #I3021 (Similar to Nonidet P-40)
FREEZER BUFFERS (-20C)
Antifade Mounting medium:
Add 1 g of, 1% p-phenylenediamine dihydrochloride (Sigma-Aldrich) to 5 ml deionized water, wrap the bottle with aluminum foil and put it on a shaker to mix for at least 2 hours. Add 5 ml 20XPBS and mix well on shaker. Add 90 ml of glycerol (Sigma-Aldrich) to the solution and leave it on shaker with gentle agitation for 30 minutes. Make 1ml aliquots of the mounting medium and store in dark at –20 oC. Take a 1 ml aliquot from –20 oC freezer and bring to room temperature before use.
100 mM sodium tetraborate buffer
Dissolve 1.9 g of sodium tetraborate decahydrate in 100 ml DEPC treated water. Adjusted pH with HCl to 8.5. The buffer was made to 1ml aliquots and stored immediately at –20oC
Denhardt’s Solution (50X)
Ficoll: 0.5g
Polyvinylpyrrlidone: 0.5g
BSA (fractionV): 0.5g
Add ddH2O to 50ml, filtered through disposable Nalgene filter and dispense into 1 ml aliquots and store at –20oC
4',6-diamidino-2-phenyindole (DAPI) solution:
Make 100 ng/ml DAPI in PBS, protect from light and keep at 4oC.
20% Bovine Serum Albumin (BSA)
BSA (Molecular grade, fraction V): 5g
Add ddH2O and adjust volume to 50 ml
Dispense into 1 ml aliquots and store at –20oC
FRIDGE BUFFERS (4C)
4',6-diamidino-2-phenyindole (DAPI) solution:
Make 100 ng/ml DAPI in PBS, protect from light and keep at 4oC.
30% Sucrose in PBS:
Dissolve 150 g sucrose in 350 ml DEPC treated water then add 25 ml 20XPBS to the solution, add DEPC water to make the total volume to 500ml. Filter the solution through 0.2 micron filter.
4% Paraformaldehyde in PBS.
CAUTION (PFA is toxic, prepare this solution in fume hood and wear protective mask while handling this solution).
Add 40g of paraformaldehyde powder to a bottle and add 700 ml of DEPC treated water. Put the bottle on a heat block and started stirring the solution with a magnetic stirrer. Monitor the temperature raise and turn off heating when the temperature reached 70oC. Continue stirring and add a few drops of 10N sodium hydroxide (NaOH) to the solution to facilitate the PFA dissolve in the water. The solution should turn clear after adding the NaOH. (CAUTION, avoid over-heat the PFA while dissolving the powder). Add 50 ml 20XPBS to the PFA solution and make up the volume to 1 liter with DEPC treated water.
10X Running buffer (WesternB)
18g Tris
86.4g Glycine
6g SDS
Up to 1l H2O
1X Running buffer (WesternB)
Take 50ml of 10X Running buffer
450ml H2O
10X Washing buffer Tween (TBST)
87.5g NaCl
15.8g Tris
Up to 1l H2O
1X Washing buffer Tween (1X TBST)
Take 50ml of 10X TBST buffer
450ml H2O
Stripping buffer (WesternB)
1.6ml of 2M tris pH 6.8
1g SDS
350ul Beta-Mercaptoethanol
Up to 50ml H2O
Dry milk solution (westernB)
2.5g dry milk
50 ml 1X TBST
WB Transfer Buffer
Glycine 14.4g
Trizma base 3.03g
Methanol 100ml
H2O 900ml
EDTA 1M
18.61g/50ml
Store at 4C