Difference between revisions of "Team:Alverno CA/Experiment"

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<h1><center>Alverno iGEM 2016</center></h1>
 
<h1><center>Alverno iGEM 2016</center></h1>
 
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<h1><center>Experiment & Protocol</center></h1>
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<h1><center>Protocol</center></h1>
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<p>      *Note: Pipettes are needed. </p>
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<h3><a href="https://2016.igem.org/Team:Alverno_CA/MakingtheAgaroseGel">Click Here to View All Protocols</a></h3>
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<h3><a href="https://2016.igem.org/Team:Alverno_CA/MakingtheAgaroseGel">Making the Agarose Gel</a></h3>
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<h3>PCR (Polymerase Chain Reaction) for Parts:</h3>
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<p>Ingredients: </p>
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<p>- 2.5μL Part Forward Primer</p>
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<p>- 2.5μL Part Reverse Primer</p>
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<p>- 0.1μL G-block / DNA template</p>
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<p>- 25μL Q5 2x High-Fidelity MasterMix</p>
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<p>- 19.9μL NFW (nuclease free water)</p>
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<p>  Materials: *</p>
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<p>- Centrifuge</p>
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<p>- Thermocycler</p>
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<p>- Mini microfuge PCR tube(s)</p>
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<p>  Directions: </p>
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<p>1. Mix above ingredients listed together in microfuge PCR tube. (Notice to add MasterMix last.)</p>
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<p>2. Spin in centrifuge.</p>
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<p>3. Put in thermocycler. Process it in thermocycler as follows: </p>
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<p>        Step 1: 98°C for 30 sec</p>
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<p>        Step 2: 98°C for 10 sec</p>
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<p>        Step 3: 70°C for 20 sec</p>
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<p>        Step 4: 72°C for 20-30sec/kilobase (typically)</p>
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<p>        Step 5: Enter “Go To” and then Step 2 and repeat for 25 cycles (or “times”)</p>
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<p>        Step 6: 72°C for 2 min</p>
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<p>        Step 7: 4°C for ∞ (Set to 00:00:00)</p>
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<p>        Step 8: End</p>
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<h3>PCR Purification of DNA: </h3>
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<p>Ingredients/Materials: *</p>
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<p>- PCR reaction(s) (DNA Part(s))</p>
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<p>- Molecular Biology Kit, which includes: </p>
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<p>      - Buffer B3 (with pre-added isopropyl alcohol)</p>
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<p>      - Wash Solution (with pre-added ethanol)</p>
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<p>      - Elution Buffer</p>
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<p>      - EZ-10 column(s)</p>
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<p>      - 1.5mL microfuge tube(s)</p>
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<p>Directions: (for each PCR reaction)</p>
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<p>    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). </p>
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<p>    2. Transfer above mixture to EZ-10 column and leave at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes. </p>
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<p>    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. </p>
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<p>    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.</p>
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<p>    5. Place top tube with white filter into clean 1.5mL microfuge tube. Check for ethanol using pipette tip. </p>
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<p>    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. </p>
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<p>    7. Store at -20 degrees Celsius, or nanodrop for concentration and for dilutions (see Parts Dilutions Protocol). </p>
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<h3>Parts Dilutions: </h3>
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<p>Ingredients/Materials: *</p>
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<p>- NFW</p>
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<p>- PCR Purified DNA Part Reaction (nanodropped with concentration)</p>
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<p>- 1.5mL microfuge tube </p>
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<p>Directions: </p>
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<p>      1. Identify number of bases and the concentration (in ng/uL, which is basically ug/mL). </p>
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<p>      2. Plug in numbers (bases and concentration) into Promega Biomath Calculator to convert from ug/mL (or ng/uL) to pmol/uL </p>
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<p>          (http://www.promega.com/a/apps/biomath/index.html?calc=ugmlpmolul).</p>
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<p>      3. Multiply resulting number by 1000 and that is the concentration in nM. </p>
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<p>      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. </p>
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<p>      5. Put in the amount of V1 of selected Part in 1.5mL microfuge tube. </p>
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<p>      6. Subtract V1 from V2. Put this amount of NFW into the tube. </p>
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<p>      7. Centrifuge.</p>
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<p>      8. Store at -20 degrees Celsius. </p>
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<h3>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)</h3>
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<p>Ingredients: (per Golden Gate Assembly)</p>
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<p>- 1μL: P part (i.e. P1a, P2a, P3a, P4a)</p>
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<p>- 1μL: UC part (i.e. UC1a, UC2a, …, UC8a, etc.) </p>
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<p>- 1μL: T part (i.e. T1a, T2a, T3a, T4a)</p>
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<p>- 1μL: V part (i.e. V19d, V1a, V2a, etc.)</p>
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<p>- 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) </p>
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<p>- 1.5μL: T4 Ligase Buffer</p>
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<p>- 0.15μL: 100x BSA Standard</p>
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<p>- 1μL: BsaI</p>
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<p>- 2μL: T4 Ligase (2M cohesive units)</p>
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<p>- 5.35μL NFW</p>
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<p>Materials: *</p>
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<p>- Centrifuge</p>
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<p>- Thermocycler</p>
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<p>- Mini microfuge tube(s)</p>
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<p>Directions: </p>
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<p>      1. Write down combos of plasmid(s) (Insert picture. 08/29/16 protocol by Melody Wu)</p>
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<p>      2. Mix above ingredients in labelled mini microfuge tube(s).</p>
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<p>      3. Spin down in centrifuge.</p>
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<p>      4. Put in thermocycler, process it in thermocycler as follows:</p>
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<p>            Step 1: 37°C for 3 min</p>
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<p>            Step 2: 16°C for 4 min</p>
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<p>            Step 3: Go to Step 1 and repeat for 25 cycles</p>
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<p>            Step 4: 50°C for 5 min</p>
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<p>            Step 5: 80°C for 5 min</p>
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<p>            Step 6: 4°C for ∞ (Set to 00:00:00)</p>
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<p>            Step 7: End</p>
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<h3>PCR Check for Golden Gate Plasmids (multiple can be done at a time in different tubes)</h3>
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<p>Ingredients: </p>
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<p>- 0.5μL V part forward sequencing primer</p>
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<p>- 0.5μL V part reverse sequencing primer</p>
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<p>- 0.1μL GG Assembly</p>
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<p>- 5μL 2x MasterMix</p>
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<p>- 3.9μL NFW</p>
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<p>Materials: *</p>
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<p>- Centrifuge</p>
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<p>- Thermocycler</p>
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<p>- Mini microfuge tube(s)</p>
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<p>Directions: </p>
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<p>      1. Mix above ingredients in mini microfuge tube(s). </p>
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<p>      2. Spin in centrifuge.</p>
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<p>      3. Put it in thermocycler, process it in thermocycler as follows: </p>
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<p>            Step 1: 98°C for 30 sec</p>
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<p>            Step 2: 98°C for 10 sec</p>
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<p>            Step 3: 56°C for 20 sec</p>
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<p>            Step 4: 72°C for 20-30sec/kilobase (typically)</p>
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<p>            Step 5: Enter “Go To” and then Step 2 and repeat for 25 cycles (or “times”)</p>
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<p>            Step 6: 72°C for 2 min</p>
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<p>            Step 7: 4°C for ∞ (Set to 00:00:00)</p>
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<p>            Step 8: End</p>
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<h3>Making LB Media (& Autoclaving)</p>
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<p>Ingredients:</p>
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<p>-LB powder
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<p>-Distilled water
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<p>Materials:</p>
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<p>-Scale</p>
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<p>-Glass bottle</p>
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<p>-Autoclave</p>
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<p>Directions:</p>
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<p>      1. Measure out 5g LB powder. </p>
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<p>      2. Add LB powder to the bottle. </p>
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<p>      3. Add 250mL distilled water to bottle. </p>
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<p>      4. Autoclave for ~30 min. </p>
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<p>      To Autoclave:
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<p>              a. Fill autoclave with water until it covers metal ring. </p>
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<p>              b. Place metal bucket in autoclave. Put in metal dish. Place bottle (or item(s) to autoclave) at center. </p>
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<p>              c. Put lid on with the dangly metal cord in place. </p>
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<p>              d. Bolt on bolts tightly to seal the lid. </p>
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<p>              e. Open both manual release valves. </p>
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<p>              f. Plug in and turn on switch. </p>
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<p>              g. Wait until steam comes out of valve and wait 5 more minutes. </p>
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<p>              h. Close both valves and wait for temperature to rise. </p>
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<p>              i. Switch off the autoclave before it reaches red at 0.15. </p>
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<p>              j. Maintain between 0.1 and 0.5 for 30 min. </p>
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<p>              k. After complete, switch off and wait until pressure goes down back to 0. </p>
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<p>              l. Open manual valves and release steam. </p>
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<h3>Making LB Agar Plates w/ Antibiotic Resistance</p>
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<p>Ingredients:</p>
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<p>- 300ml H2O</p>
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<p>- 6g LB Powder</p>
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<p>- 4.5g Agar Powder</p>
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<p>- Antibiotic (usually use Kanamycin - 1μL per 1mL H2O)</p>
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<p>Materials: *</p>
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<p>- Autoclave</p>
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<p>- Scale</p>
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<p>- Glass bottle</p>
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<p>Directions: </p>
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<p>      1. Measure out the LB and Agar. Pour into a glass bottle and then pour in distilled water up to 300mL line. </p>
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<p>      2. Mix the above ingredients. </p>
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<p>      3. Autoclave for 30 minutes. </p>
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<p>      4. Cool down to around 50°C (~122°F). </p>
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<p>      5. Add 300μL Kanamycin (or chosen antibiotic, added accordingly).</p>
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<p>      6. Open lid to each plate carefully and pour plate near flame. </p>
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<h3>Bacterial Transformation of Plasmids (& Growing Liquid Cultures) </p>
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<p>Ingredients/Materials: *</p>
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<p>- DNA GG Plasmid Mixture</p>
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<p>- Competent Cells</p>
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<p>- SOC Media (or LB Media if SOC is contaminated…) </p>
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<p>- 2ml Microtubes</p>
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<p>- Tube Rack</p>
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<p>- Ice</p>
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<p>- Timer</p>
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<p>- 42°C Water Bath (set early on!) </p>
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<p>- 37°C Incubator (set early on!) </p>
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<p>- Petri Plates with LB agar and antibiotic</p>
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<p>- Sterile Spreader or sterile glass beads</p>
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<p>Directions: </p>
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<p>      1. Thaw competent cells on ice. (If delicate, take out after step 3, and do step 2 after step 3) </p>
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<p>      2. Pipette 20μL of competent cells into 2mL tube.</p>
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<p>      3. Pipette 1μL of control DNA into 2mL tube.</p>
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<p>      4. Pipette 1μL of resuspended DNA into 2mL tube.</p>
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<p>      5. Close 2ml tubes and incubate on ice for 30 minutes. </p>
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<p>      6. Heat shock tubes at 42°C for 1 minute.</p>
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<p>      7. Incubate on ice for 5 minutes. </p>
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<p>      8. Pipette 200μL SOC media (or LB media) to each transformation.</p>
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<p>      9.  Incubate at 37°C for 2 hours—in incubator. </p>
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<p>      10. Pipette each transformation on petri plates (labelled!).</p>
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<p>      11. Incubate transformations overnight (14-18 hours) at 37°C. </p>
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<p>  Next Day: </p>
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<p>      12. Pick single colonies. Transfer each single colony to a gridded plate (labelled), dip pipette tip into PCR reaction to do a colony PCR to verify part size, then place tip into liquid culture to grow up liquid cell cultures. </p>
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<p>      13. To do Colony PCR, create mixture according to PCR Check for Golden Gate Plasmids Protocol without 0.1uL of GG Assembly (or DNA Plasmid). Put in the MasterMix as well.
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</p>
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<p>      14. After picking colony with pipette tip dip into reaction mixture and then put PCR reaction into the thermocycler and continue PCR protocol. </p>
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<p>      15. Incubate gridded plate and the liquid cultures at 37°C overnight. Take out the next morning and store in refrigerator (4°C). </p>
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<h3>Plate Reading (for Fluorescence, Absorbance, Induction, etc.)
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<p>Ingredients/Materials: *
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<p>- Liquid cultures
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<p>- 96 well plate (A-H by 1-12)
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<p>- Plate Reader (we use VICTOR X3)
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<p>- LB Media
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<p>Directions:
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<p>      1. Pipette in 100uL per well for each liquid culture. Place plate into reader. Set protocol to absorbance and hit run (green arrow button).
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<p>      2. Measure Absorbance at 600nm for all samples in all standard measurement modes in plate reader.
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<p>      3. Import data into Excel Sheet after run is finished. Use Normalization sheet tab and copy over. Enter data accordingly.
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<p>      4. Record the data, specifically volume of preloading culture and preloading media from the table in the notebook.
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<p>      5. Dilute accordingly (media is LB media with antibiotic) in a new 96 well plate (if needed). Usually 500uL per well.
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<p>      6. Place plate back into reader and set to OD-RFP-GFP protocol and start run.
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<p>      7. Let it run overnight (120 runs total with about 30 second intervals) and check in the morning.
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<p>      8. Import data results into Excel spreadsheet. Upload to Google drive.
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<p>      9. To analyze data using Python program—file must be in csv format. (If you would like the code for analyzing this type of data, please contact us!)
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<p>For any questions about Protocols, email: alverno.igem@gmail.com</p>
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Revision as of 22:11, 6 October 2016

Alverno iGEM 2016

Alverno iGEM 2016

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