Miscellaneous Recipes
RNase
- Dissolve pancreatic RNase (RNase A) at 10 mg/mL in
- 150 uL 1 M Tris (pH 7.5)
- 45 uL 5 M NaCl
- q.s. to 15 mL w/ ddH2O
- Boil 15 minutes; allow to cool to room temp.
- Dispense into aliquots and store at -20oC
Trypsin w/ 0.1 mM EDTA/PBS
- 25 mL 10x Trypsin
- 50 uL 0.5 M EDTA
- 25 mL 10x PBS
- 200 mL ddH2O
- Filter sterilize; store at 4oC.
1x PBS (for Tissue Culture)
- 10 mL 10x PBS
- 90 mL ddH2O
- Filter sterilize; store at 4oC.
10x Ligation Buffer
[Final]
- 0.5 M Tris, pH 7.5
- 100 mM MgCl2
- 100 mM DTT
- 0.5 mg/mL BSA
- 10 mM ATP
- 5 mM Spermidine
BSA 10x Buffer for DRA III
- 10 uL BSA
- 90 uL ddH2O
- Vortex; place 1 uL per sample.
Ear Punch Digestion PCR Buffer
- Have 10x PCR buffer stock in 1 mL aliquots
- Add 1 mL 10x PCR buffer to 9 mL ddH2O mix by inverting. (now 1x solution)
- Add 45 uL Triton x-100
- Add 10 uL Polyoxyethylene Sorbitan Monolaurate (Tweene 20)
- Vortex.
- Add 200 uL to each ear punch.
Polylysine
- Stock 1 mg/mL
- Use 100 ug/mL
- Filter sterilize solutions.
- Take bottle of Poly-L-Lysine and dissolve in ½ bottle of H2O then q.s. to 100 mL.
- Filter sterilize 10x solution of 1 mg/mL
- Then take 10 mL of stock solution + 90 mL H2O
- Filter sterilize for using/working solution.
Tunicamycin
- Stock 100 ug/mL final (1000x)
- 1 mg in 10 mL H2O(for immediate use)
- Filter sterilize; store at -20oC.
Calcium Chloride (50 mM)
0.736 g. CaCl2*2H2O
10 mL 1 M Tris-Cl pH 8.0
90 mL H2O
Filter Sterilize; store at 4oC.
GIEMSA Stain*
For 100 mL:
20 mL GIEMSA
80 mL Methanol
*stains permanently! Use gloves and lab coat.
Lysis Buffer
- 20 mL Buffer A
- 40 uL 1 M MgCl2
- 100 uL Triton x-100
- 200 uL Aprotonin
- 10 uL Leupeptin (10 ug/mL stock)
MAP Kinase Buffer
Buffer A:
- 15.3 g. B-glycerophosphate
- 4 mL 0.25 M EGTA (Ethylene Glycol Tetraacetic Acid)
- 0.5 mL 200 mM Na3VO4
- 154 mg DTT
- Adjust pH to 7.2; q.s. to 1000 mL w/ ddH2O
- Filter into fresh bottles.
Sodium Hydroxide, 0.2 M
- 0.08 g. NaOH
- 10 mL H2O
BSA, 1 mg/mL (Bovine Serum Albumin)
10 mg BSA
10 mL ddH2O
Freeze Media
- 8 mL DMEM w/ 10% FBS
- 1 mL FBS
- 1 mL DMSO
F-12 Media
For 1 Liter:
- 5 g. Glucose (powder)
- 10.6 g. F12
- 1.167 NaHCO3
- ~950 mL ddH2O
- adjust pH to 7.15
- q.s. to 1 L w/ ddH2O
For 4 Liters:
- 20.0 g. Glucose (powder)
- 42.4 F12
- 4.7 g. NaHCO3
- 3800 mL ddH2O
- Adjust to pH 7.15
- q.s. to 4 L.
DMEM for Tissue Culture:
*Make 16-20 L at a time.
For 4 L:
- 53.93 g. DME
- 14.8 g. NaHCO3
- 3800 mL ddH2O
- Adjust to pH 7.15
- q.s. to 4 L.
- Filter sterilize, store at 4oC.
G418 Selection Media:
- 900 mL DME
- 100 mL FBS
- 10 mL Pen/Strep
- 15 mL G418 (want concentration to = 750 ug/mL)
G418 Stock Solution
- Called Geneticin G-418 Sulfate
- Want stock to be 50 mg/mL
- Add full bottle of G418 and 100 mL ddH2O
- Filter sterilize in hood in 150 mL Nalgene filter
- Aliquot into 10 mL conical vials.
- 7.5 % NCS + 2.5% FBS
- 750 mL NCS
- 25 mL FBS
- 10 mL Pen/Strep
- Polybrene (Hexadimethrine Bromide)
- 8 mg/mL (1000x) stock
- 80 mg in 10 mL ddH2O.
- Filter sterilize; store at -20oC.
10X SAB Buffer
For 500 mL
- 33.32 g NaCl
- 1.73 g KCl
- 0.82 g KH2PO4
- 0.70 g MgSO4
- q.s. to 500 mL w/ddH2O
- Filter sterilize
For 1000 mL
- 66.64 g NaCl
- 3.46 g KCl
- 1.64 g KH2PO4
- 1.40 g MgSO4q.s. to 1000 mL w/ddH2O
- Filter sterilize
1x SAB
For 100 mL
- 10 mL 10x SAB
- 2 mlL1M HEPES .57 mL 35% BSA
- .21 g NaHCO3 q.s. to 90 mL w/ ddH2O
- 1 mL .25 M CaCl2
- 100 uL 45% glucose
- q.s. 100 mL dd H2O
***HEPES, BSA, and glucose should be added in the hood.
***If it needs to be used long term (more than that day) it should be sterile filtered.
***You can add glucose as it is used to help reduce contamination risks.