Difference between revisions of "Team:Hong Kong HKU/Experiments"

General preparation of Native Polyacrylamide gel (Native PAGE gel)

Specifications
DNA working solution (1μM)
Requiring Materials

Materials	Quantity
DNA Stock solution (100μM)	5.0μL/5.5μL
1X TM buffer	1 Set

Storage
-20°C
Steps
1. Wipe the bench and gloves with 70% ethanol.
2. Briefly vortex the stock solutions before dilution.
3. Prepare the followings.

Eppendorf/PCR tube Label	Volume of 1X TM buffer	Volume of 100μM stock solution	Final volume and concentration
O1 (5μM)	95 μL	5 μL	5 μM 100 μL
O2 (5μM)	95 μL	5 μL	5 μM 100 μL
O3 (5μM)	95 μL	5 μL	5 μM 100 μL
O4 (5μM)	95 μL	5 μL	5 μM 100 μL
O5 (5μM)	95 μL	5 μL	5 μM 100 μL
O1 (25μM)	16.5 μL	5.5 μL	25 μM 22 μL
O2 (25μM)	16.5 μL	5.5 μL	25 μM 22 μL
O3 (25μM)	16.5 μL	5.5 μL	25 μM 22 μL
O4 (25μM)	16.5 μL	5.5 μL	25 μM 22 μL
O5 (25μM)	16.5 μL	5.5 μL	25 μM 22 μL
Input (5μM)	95 μL	5 μL	5 μM 100 μL

4. Mix well by tapping followed by briefly centrifuging the eppendorfs.
Notes
TM Buffer is a Tris buffer containing Magnesium ion. Here, we prepare TM Buffer of 40 mM Tris, 100mM MgCl2 and is maintained at pH 8.0 condition.
Refer to the following for the preparation of TM buffer:

Materials	Quantity
MgCl2·2H2O or MgCl2 or MgCl2·6H2O	0.566g or 0.476g or 1.016g
Distilled water	48 mL
1M Tris solution	2 mL

For the steps, it is noted that the pH of the buffer is adjusted to 8 by adding HCl with the aid of a pH meter. The above quantity is in 2X concentarion, therefore the solution is further diluted to 1X by mixing 115μL 2X TM buffer with 115μL ddH2O to an eppendorf.

Tetrahedron assembly and preparation of DNA solutions for PAGE

General preparation of Native Polyacrylamide gel (Native PAGE gel)

Specifications
A piece of PAGE Gel (~12mL in volume)
Requiring Materials

Materials (12% PAGE gel)	Quantity
Gel kit	1 Set
Distilled water	6.0 mL
10X TBE	1.2 mL
30% Acrylamide (29:1)	4.8 mL
10% APS	200 μL
TEMED	200 μL

Storage
4°C, keep wet in 1x TBE to prevent drying
Steps
1. Clean the glass plates and spacers thoroughly. Rinse the plates withdeionized water and ethanol and set them aside to dry.
2. Assemble the glass plates with spacers in gel caster. Make sure there is no water leakage.
3. Prepare the gel solution according to the desired polyacrylamide percentage. Note the addition of TEMED will immediately trigger the gel to polymerize.
4. Vortex the solution roughly for 5 seconds.
5. Quickly piette the gel solution into the caster. Insert the appropriate comb into the gel, prevent to trap air bubbles under the teeth.
6. Let the polymerization go for 30 minutes at room temperature.
7. Surround the comb and the top of the gel with paper towels that have been soaked in 1x TBE. Then seal the entire gel in plastic bag and store it at 4°C until needed.
Notes
Different polyacrylamide percentage results in different speed and discrimating ability for sample running down in electrophoresis. The quantity of the solutions varies with the percentage. The required quantity of native PAGE gel in common polyacrylamide percentage is listed below.

Polyacrylamide percentage	Distilled water	30% Acrylamide (29:1)	10X TBE	10% APS	TEMED
8%	7.6 mL	3.2 mL	1.2 mL	200 μL	10 μL
10%	6.8 mL	4.0mL	1.2 mL	200 μL	10 μL
12%	6.0 mL	4.8 mL	1.2 mL	200 μL	10 μL
15%	5.2 mL	5.6 mL	1.2 mL	200 μL	10 μL

Native PAGE

Steps
Load the followings. (DNA ladder: 2μL, DNA sample: 8μL, 1X loading dye: 8μL)

Lane	1	2	3	4	5	6	7	8	9	10
Gel A	20 bp DNA ladder	O1.6	O2	O3	O4	O5	Input	A1	A2	A3
Gel B	20 bp DNA ladder	A4	A5	A6	A7	A8	A9	A10	B1	B2
Gel C	20 bp DNA ladder	B3	B4	B5	B6	B7	B8	B9	B10	1X Loading buffer
Gel D	20 bp DNA ladder	C1	C2	C3	C4	C5	Tetra	Tetra+Input	Output	1X Loading buffer

Run the gel at constant voltage at 100V for until the bands of dye reach ¾ of the length of the gel.