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
PCR Purification of DNA:
Ingredients/Materials: *
- PCR reaction(s) (DNA Part(s))
- Molecular Biology Kit, which includes:
- Buffer B3 (with pre-added isopropyl alcohol)
- Wash Solution (with pre-added ethanol)
- Elution Buffer
- EZ-10 column(s)
- 1.5mL microfuge tube(s)
Directions: (for each PCR reaction)
1. Transfer PCR reaction mixture (usually 50ul, ranges from 35-50ul) to a 1.5mL microfuge tube and add 5 volumes (5 x amount of PCR reaction mixture) of Buffer B3 (with pre-added isopropyl alcohol).
2. Transfer above mixture to EZ-10 column and leave at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes.
3. Remove/empty flow-through in bottom tube. Add 750ul of Wash Solution (with pre-added ethanol) and centrifuge at 10,000rpm for 2 minutes.
4. Repeat washing procedure (from Step 3, “Add 750ul of…”). Remove/empty flow-through again. Spin at 10,000rpm for an additional minute. Throw away bottom clear tube with any remaining liquid.
5. Place top tube with white filter into clean 1.5mL microfuge tube. Check for ethanol using pipette tip.
6. Add 30-50uL (usually 40uL) of Elution Buffer to center of tube. Incubate at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes to elute DNA.
7. Store at -20 degrees Celsius, or nanodrop for concentration and for dilutions (see Parts Dilutions Protocol).
Parts Dilutions:
Ingredients/Materials: *
- NFW
- PCR Purified DNA Part Reaction (nanodropped with concentration)
- 1.5mL microfuge tube
Directions:
1. Identify number of bases and the concentration (in ng/uL, which is basically ug/mL).
2. Plug in numbers (bases and concentration) into Promega Biomath Calculator to convert from ug/mL (or ng/uL) to pmol/uL
(http://www.promega.com/a/apps/biomath/index.html?calc=ugmlpmolul).
3. Multiply resulting number by 1000 and that is the concentration in nM.
4. Plug into dilution equation: C1*V1=(30nM)(V2), where C1 is the concentration in nM, and V2 is equal to the amount wanted (typically 10uL-20uL). Then solve for V1.
5. Put in the amount of V1 of selected Part in 1.5mL microfuge tube.
6. Subtract V1 from V2. Put this amount of NFW into the tube.
7. Centrifuge.
8. Store at -20 degrees Celsius.
Golden Gate Assembly for Plasmids (Example with Golden Gate Assembly Protocol for GG37-52; multiple GG Assembly for Plasmids can be done at a time in different tubes as seen in example)
Ingredients: (per Golden Gate Assembly)
- 1μL: P part (i.e. P1a, P2a, P3a, P4a)
- 1μL: UC part (i.e. UC1a, UC2a, …, UC8a, etc.)
- 1μL: T part (i.e. T1a, T2a, T3a, T4a)
- 1μL: V part (i.e. V19d, V1a, V2a, etc.)
- 1μL: GFP Mut Parts: P1ab, P2ab, T3ab, T4ab (Note: for all parts with GFP, matches according to Part, see combos image for example; some do not have GFP Mut parts and so add 1ul to NFW amount instead)
- 1.5μL: T4 Ligase Buffer
- 0.15μL: 100x BSA Standard
- 1μL: BsaI
- 2μL: T4 Ligase (2M cohesive units)
- 5.35μL NFW
Materials: *
- Centrifuge
- Thermocycler
- Mini microfuge tube(s)
Directions:
1. Write down combos of plasmid(s) (Insert picture. 08/29/16 protocol by Melody Wu)
2. Mix above ingredients in labelled mini microfuge tube(s).
3. Spin down in centrifuge.
4. Put in thermocycler, process it in thermocycler as follows:
- Step 1: 37°C for 3 min
- Step 2: 16°C for 4 min
- Step 3: Go to Step 1 and repeat for 25 cycles
- Step 4: 50°C for 5 min
- Step 5: 80°C for 5 min
- Step 6: 4°C for ∞ (Set to 00:00:00)
- Step 7: End
