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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 KH​2​PO​4
0.70 g MgSO​4
q.s. to 500 mL w/ddH2O
Filter sterilize
For 1000 mL
66.64 g NaCl
3.46 g KCl
1.64 g KH​2​PO​4
1.40 g MgSO​4
q.s. to 1000 mL w/ddH​2​O
Filter sterilize