C
Carbonate/Bicarbonate buffer
200 ml 1 L .
Na2CO3 0.3 g 1.5 g (14 mM)
NaHCO3 0.6 g 3.0 g (36 mM)
MgCl2:6H2O 0.2 g 1.0 g (5 mM)
pH to 9.8
QS to vol
Cell lysis buffer
Includes protease inhibitors
200 µl PMSF
10 µl Aprotinin (10 mg/ml stock)
10 µl Leupeptin (10 mg/ml stock)
500 µl Triton X-100
500 µl 1M Tris-Cl
1000 µl 3M NaCl
7780 µl PBS (cold)
CIAP (dephosphorylation to prevent cut vectors from re-ligating)
Catalyzes the removal of 5´ phosphate groups from DNA, RNA, ribo- and deoxyribonucleoside triphosphates.
-Dilute CIAP (Calf intestine alkaline phosphatase) 1:10 using Dilution Buffer (provided with kit)
-If RXN mix is going to be at a final volume of 50 µl then:
-43 µl sample (e.g., cut PQE-30 [Qiagen])
-2 µl Diluted CIAP
-5 µl 10X dephosphorylation buffer
-30 min @ 37°C followed by 10 min @ 68°C.
Charge buffer (8X) (see IDA columns; Charge buffer not needed for already pre-charged columns)
14.28 g NiCl2:6H2O (600 mM)
QS to 100 ml with H2O.
Sterile filter.
Complete CTL (T-cell growth media)
5 ml Non essential amino acids (100X stock)
5 ml Sodium bicarbonate (7% aqueous solution stock)
5 ml Sodium pyruvate (100 mM stock)
2.5 ml HEPES (1 M stock)
5 ml L-glutamine (200 mM stock)
5 ml Pen-Strep (100X stock)
0.50 ml 2 ME (5 mM stock) (Final concentration of 0.055 mM) Must be fresh (VERY IMPORTANT!)
5 ml Vitamin solution (100X stock)
50 ml 'gold' FBS
0.5 ml gentamycin (50 mg/ml stock)
QS to 500 ml with RPMI 1640.
Sterile filter
D
DB8 10X
Tris HCL 44.4 g
Tris Base 26.5 g
Glycine 75.07 g
pH to 8.0
QS to 1 L
DEA (Diethanolamine)
1 L final vol
1.3 M DEA (pH 9.8) 124 ml (DEA)
0.5 mK MgCl2 500 ul
H20 QS to volume
pH before adding the MgCl2
to use, dissolve 1 tablet (5 mg) Sigma 104 phosphatase in 5 ml DEA.
-Light sensitive. Prepare immediately before use.
Decalcification (histology)
Any samples containing bone must be de-calcified prior to embedding and sectioning.
Incubate samples in 5% Formic Acid (88%) for at least 3 days.
Destain (SDS-PAGE)
200 ml (final vol) 1 L (final vol)
50% Methanol 100 ml 500 ml
10% Acetic acid 20 ml 100 ml
ddH20 80 ml 400 ml
Destain (Nitrocellulose membranes)
10% Methanol
10% Glacial Acetic acid
80% ddH20
DNA concentration determination
A260 X 50 X dilution = µl/ml
DNA Markers
25 µl DNA
782.1 µl H20
41.25 µl 1M NaCl (50 mM final)
1.65 µl 0.5 M EDTA (1 mM final)
150 µl 6X Blue Juice
DTT (dithiothreitol, 154.2 M.W.)
-Dissolve 3.09 g DTT in 20 ml of 0.01 M Sodium Acetate (pH 5.2).
-Sterile filter.
-Dispense into 1 ml aliquotes and store at -20°C.
Carbonate/Bicarbonate buffer
200 ml 1 L .
Na2CO3 0.3 g 1.5 g (14 mM)
NaHCO3 0.6 g 3.0 g (36 mM)
MgCl2:6H2O 0.2 g 1.0 g (5 mM)
pH to 9.8
QS to vol
Cell lysis buffer
Includes protease inhibitors
200 µl PMSF
10 µl Aprotinin (10 mg/ml stock)
10 µl Leupeptin (10 mg/ml stock)
500 µl Triton X-100
500 µl 1M Tris-Cl
1000 µl 3M NaCl
7780 µl PBS (cold)
CIAP (dephosphorylation to prevent cut vectors from re-ligating)
Catalyzes the removal of 5´ phosphate groups from DNA, RNA, ribo- and deoxyribonucleoside triphosphates.
-Dilute CIAP (Calf intestine alkaline phosphatase) 1:10 using Dilution Buffer (provided with kit)
-If RXN mix is going to be at a final volume of 50 µl then:
-43 µl sample (e.g., cut PQE-30 [Qiagen])
-2 µl Diluted CIAP
-5 µl 10X dephosphorylation buffer
-30 min @ 37°C followed by 10 min @ 68°C.
Charge buffer (8X) (see IDA columns; Charge buffer not needed for already pre-charged columns)
14.28 g NiCl2:6H2O (600 mM)
QS to 100 ml with H2O.
Sterile filter.
Complete CTL (T-cell growth media)
5 ml Non essential amino acids (100X stock)
5 ml Sodium bicarbonate (7% aqueous solution stock)
5 ml Sodium pyruvate (100 mM stock)
2.5 ml HEPES (1 M stock)
5 ml L-glutamine (200 mM stock)
5 ml Pen-Strep (100X stock)
0.50 ml 2 ME (5 mM stock) (Final concentration of 0.055 mM) Must be fresh (VERY IMPORTANT!)
5 ml Vitamin solution (100X stock)
50 ml 'gold' FBS
0.5 ml gentamycin (50 mg/ml stock)
QS to 500 ml with RPMI 1640.
Sterile filter
D
DB8 10X
Tris HCL 44.4 g
Tris Base 26.5 g
Glycine 75.07 g
pH to 8.0
QS to 1 L
DEA (Diethanolamine)
1 L final vol
1.3 M DEA (pH 9.8) 124 ml (DEA)
0.5 mK MgCl2 500 ul
H20 QS to volume
pH before adding the MgCl2
to use, dissolve 1 tablet (5 mg) Sigma 104 phosphatase in 5 ml DEA.
-Light sensitive. Prepare immediately before use.
Decalcification (histology)
Any samples containing bone must be de-calcified prior to embedding and sectioning.
Incubate samples in 5% Formic Acid (88%) for at least 3 days.
Destain (SDS-PAGE)
200 ml (final vol) 1 L (final vol)
50% Methanol 100 ml 500 ml
10% Acetic acid 20 ml 100 ml
ddH20 80 ml 400 ml
Destain (Nitrocellulose membranes)
10% Methanol
10% Glacial Acetic acid
80% ddH20
DNA concentration determination
A260 X 50 X dilution = µl/ml
DNA Markers
25 µl DNA
782.1 µl H20
41.25 µl 1M NaCl (50 mM final)
1.65 µl 0.5 M EDTA (1 mM final)
150 µl 6X Blue Juice
DTT (dithiothreitol, 154.2 M.W.)
-Dissolve 3.09 g DTT in 20 ml of 0.01 M Sodium Acetate (pH 5.2).
-Sterile filter.
-Dispense into 1 ml aliquotes and store at -20°C.
DNA Extraction (from mammalian tissue) (Example: mouse tails)
-Cut tip of tail and place in 2 ml epitube; add 500 ul Lysis buffer.
-Lysis buffer (0.05 M EDTA, 0.5 g SDS, 0.05 M Tris, 0.1 M NaCl, 0.005 M DTT, 0.0005 spermidine.
-After adding lysis buffer add 0.4 mg/ml Protinase K (stock at 20 mg/ml).
-Rock E/E @ 55-60°C o/n.
-Pellet debris by centrifugation for 4 min at 13,000 rpm.
-Transfer supernatant to a new tube and add 1 ml of 100% EtOH.
-Invert tubes gently.
-Spool DNA with sterile pipet and rinse with 70% EtOH.
-Transfer DNA to a new tube and allow to dry very well. No traces of EtOH must remain.
-Add 500 µl sterile autoclaved H2O. Rock o/n @ RT.
-Store at -70°C until use.
-Cut tip of tail and place in 2 ml epitube; add 500 ul Lysis buffer.
-Lysis buffer (0.05 M EDTA, 0.5 g SDS, 0.05 M Tris, 0.1 M NaCl, 0.005 M DTT, 0.0005 spermidine.
-After adding lysis buffer add 0.4 mg/ml Protinase K (stock at 20 mg/ml).
-Rock E/E @ 55-60°C o/n.
-Pellet debris by centrifugation for 4 min at 13,000 rpm.
-Transfer supernatant to a new tube and add 1 ml of 100% EtOH.
-Invert tubes gently.
-Spool DNA with sterile pipet and rinse with 70% EtOH.
-Transfer DNA to a new tube and allow to dry very well. No traces of EtOH must remain.
-Add 500 µl sterile autoclaved H2O. Rock o/n @ RT.
-Store at -70°C until use.
Competent Cells
-Inoculate 100 µl of o/n culture of cells into
25 ml LB and incubate @ 37°C for 5 h.
-Place 2m ml tube on ice for 5 min.
-Spin for 5 min @ 2400 RPM; remove
supernatant.
-Resuspend pelet in 8.3 ml of RF1, incubate
on ice for 20 min.
Spin 5 min @ 2400 RPM; remove
supernatant.
-Resuspend pellet in 2 ml RF2 and incubate
on ice for 20 min.
Aliquote 200 µl of 'competent' cells @ -70°C.
-Freeze down by placing epitubes in dry ice
containing EtOH (snap freezing); then
move to -70°C.
-Inoculate 100 µl of o/n culture of cells into
25 ml LB and incubate @ 37°C for 5 h.
-Place 2m ml tube on ice for 5 min.
-Spin for 5 min @ 2400 RPM; remove
supernatant.
-Resuspend pelet in 8.3 ml of RF1, incubate
on ice for 20 min.
Spin 5 min @ 2400 RPM; remove
supernatant.
-Resuspend pellet in 2 ml RF2 and incubate
on ice for 20 min.
Aliquote 200 µl of 'competent' cells @ -70°C.
-Freeze down by placing epitubes in dry ice
containing EtOH (snap freezing); then
move to -70°C.
Coomassie Stain
Percent Amount
R250 Coommassie 0.25% 2.5g
MeOH 50% 500 ml
Acetic Acid 7% 70 ml
ddH2O QS to 1 L
Percent Amount
R250 Coommassie 0.25% 2.5g
MeOH 50% 500 ml
Acetic Acid 7% 70 ml
ddH2O QS to 1 L
Chimicuri (Lets face it; if you can't cook get out of the lab! Besides, this is a good recipe!)
1 bunch chopped parsley
5 chopped garlic cloves
1/4 teaspoon fresh ground black pepper
1 teaspoon NaCl
1-tablespoon oregano
1 tablespoon red pepper flakes
1/2 cup olive oil
1/4 cup canola oil
1/2 cup apple cider vinegar)
1 bunch chopped parsley
5 chopped garlic cloves
1/4 teaspoon fresh ground black pepper
1 teaspoon NaCl
1-tablespoon oregano
1 tablespoon red pepper flakes
1/2 cup olive oil
1/4 cup canola oil
1/2 cup apple cider vinegar)