Team:Alverno CA/Experiment

Alverno iGEM 2016

Experiment & Protocol

*Note: Pipettes are needed.

Making the Agarose Gel:

Ingredients/Materials: *

- 0.6g agarose

- 50mL TBE 1x

- 5μL SYBRsafe / 2.5μL EcoStain (light- and heat-sensitive)

- 250mL conical flask

- scale

- Microwave

- Gel mold

Directions:

1. Weigh 0.6g of agarose into flask.

2. Add 50mL of TBE 1x Buffer

3. Microwave agarose solution until dissolved for 1 min (take out halfway to swirl)

(If the liquid looks distorted, agarose solution need to be microwaved more)

4. Cool slightly and add SYBRsafe or EcoStain

5. Pour gel into mold and put the combs into their spots

6. Cool until solidified.

Gel Electrophoresis & Screening the gel

Ingredients/Materials: *

- TBE 1x Buffer

- agarose gel (w/ right number of wells)

- DNA reaction (either parts=PUCTV, or PCR check for GG plasmids)

- purple loading dye

- Gel box

Directions:

1. Place agarose gel into gel box with wells on negative side.

2. Fill up gel box up with TBE 1x Buffer up to line or at least above gel.

3. Pipette 6ul of 2-log ladder on side lanes (or given wells).

4. Pipette 1ul of purple loading dye on parafilm. Then pipette 5ul of DNA reaction and mix with loading dye on parafilm. (*For PCR Check: Directly pipette 2ul of purple loading dye into 10ul of PCR Check Reaction in microfuge tube)

5. Set to 6ul and pipette 6ul of mixed reaction with loading dye into selected wells according to drawn diagram. See gel picture.

6. Set machine to 175V and for 20 minutes. Make sure bubbles are appearing!

7. After running place gel onto transilluminator (must be off) in glove box. Place box over it with hole and then place iPad above it and set to either video or time-lapse. Then turn on transilluminator within closed glove box while videotaping.

8. Results can now be analyzed.

PCR (Polymerase Chain Reaction) for Parts:

Ingredients:

- 2.5μL Part Forward Primer

- 2.5μL Part Reverse Primer

- 0.1μL G-block / DNA template

- 25μL Q5 2x High-Fidelity MasterMix

- 19.9μL NFW (nuclease free water)

Materials: *

- Centrifuge

- Thermocycler

- Mini microfuge PCR tube(s)

Directions:

1. Mix above ingredients listed together in microfuge PCR tube. (Notice to add MasterMix last.)

2. Spin in centrifuge.

3. Put in thermocycler. Process it in thermocycler as follows:

a. Step 1: 98°C for 30 sec

b. Step 2: 98°C for 10 sec

c. Step 3: 70°C for 20 sec

d. Step 4: 72°C for 20-30sec/kilobase (typically)

e. Step 5: Enter “Go To” and then Step 2 and repeat for 25 cycles (or “times”)

f. Step 6: 72°C for 2 min

g.Step 7: 4°C for ∞ (Set to 00:00:00)

h. Step 8: End