Difference between revisions of "Team:CSU Fort Collins/Experiments"
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− | <div class="flip" id="flip1" href ="#panel1"> <h4>Terpenoid | + | <div class="flip" id="flip1" href ="#panel1"> <h4>Terpenoid Growth Curve</h4> |
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<h3>Materials</h3> | <h3>Materials</h3> | ||
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+ | <p></p> | ||
+ | <div class="flip" id="flip1" href ="#panel1"> <h4>Terpenoid Extraction and Detection</h4> | ||
+ | <div class="panel" id="panel1"> | ||
+ | <h3>Materials</h3> | ||
+ | <ul> <li>First</li> | ||
+ | <li>Second</li> | ||
+ | <li>Third</li> | ||
+ | </ul> | ||
+ | <h3 >Procedure</h3> | ||
+ | <ol> <li>First</li> | ||
+ | <li>Second</li> | ||
+ | <li>Third</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
<p></p> | <p></p> | ||
Revision as of 16:08, 7 September 2015
Click the panels to slide down or up
Protocols
PCR
Piece Amplification
Component | 50μl reaction |
---|---|
Molecular grade H2O | Added first. 32.5μL |
5x HF Buffer | 10μL |
10 mM dNTPs | 1.0μL |
Primer A (10μM) | 2.5μL |
Primer B (10μM) | 2.5μL |
Template DNA | 1.0μL |
DNA polymerase | Added last. 0.5μL |
Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
Cycle Step | Temp(C) | Time | Cycles |
---|---|---|---|
Initial denaturation | 98 | 5 min | 1 |
Denaturation | 98 | 10s | 35 |
Annealing | LowerTm+3 | 15s | 35 |
Extension | 72 | 45s/kb | 35 |
Final Extension | 72 | 10min | 1 |
Hold | 4 | Hold | 1 |
Component | 50μl reaction |
---|---|
Molecular grade H2O | Added first 33.5ul |
5x Phusion HF Buffer | 10μL |
10 mM dNTPs | 1.0μL |
Primer A (10μM) | 2.5μL |
Primer B (10μM) | 2.5μL |
Colony | 1 |
Phusion DNA polymerase | Added last. 0.5μL |
Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
Cycle Step | Temp(C) | Time | Cycles |
---|---|---|---|
Initial denaturation | 98 | 5 min | 1 |
Denaturation | 98 | 10s | 35 |
Annealing | LowerTm+3 | 15s | 35 |
Extension | 72 | 45s/kb | 35 |
Final Extension | 72 | 10min | 1 |
Hold | 4 | Hold | 1 |
Digestion
Component | 50μl reaction |
---|---|
Molecular Grade Water | Calculated. Use to make reaction total volume = 50ul |
Custsmart Buffer(different if using pstI) | 5ul |
DNA | 1ug |
Enzyme 1 | .5ul |
Enzyme 2 | 0.5ul |
Procedure
Digest your mixture in a water bath at 37C for 60min
Ligation
Component | 20μl reaction |
---|---|
Molecular Grade Water | Calculated. Use to make reaction total volume = 20ul |
T4 DNA Ligase Buffer | 2ul |
Insert DNA | |
Backbone DNA | |
T4 DNA Ligase | 1ul |
Incubate at 16C overnight on a heating block
Transformation
Plasmid Construction
What is the template for your PCR:
Primer A:_________ Tm=__C
Primer B:_________ Tm=__C
Component | 50μl reaction |
---|---|
Molecular grade H2O | Added first. 32.5μL 33.5ul for colony PCR |
5x Phusion HF Buffer | 10μL |
10 mM dNTPs | 1.0μL |
Primer A (10μM) | 2.5μL |
Primer B (10μM) | 2.5μL |
Template DNA | 1.0μL 0uLcolony PCR |
Phusion DNA polymerase | Added last. 0.5μL |
Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
Cycle Step | Temp(C) | Time | Cycles |
---|---|---|---|
Initial denaturation | 98 | 5 min | 1 |
Denaturation | 98 | 10s | 35 |
Annealing | LowerTm+3 | 15s | 35 |
Extension | 72 | 45s/kb | 35 |
Final Extension | 72 | 10min | 1 |
Hold | 4 | Hold | 1 |
- Measure concentration of clean-up PCR products with a nano drop
- Run 10µL of clean-up PCR with 2ul of SybrGreen DMSO products on a gel 1%Agarose Gel (110V for 60mins). Look for How Many Base Pairs your insert is
- Digest (Insert) and (Backbone )with ________+__________ at 37oC for 90mins.
- Clean up all DNA fragments with PCR clean-up kit.
- Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1
- Incubate at 16C overnight on a heating block.
- Transformation (Need 2x LB+Antibiotic plates. Check for plates. Check for hockey spreaders or glass beads):
- Set water bath to 42C
- Remove LB+Antibiotic plates from 4C and allow them to come to RT.
- Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
- Add 5μL of ligation mix chemically competent cells.
- Add 5uL of digested back bone to chemical competent cells as a control
- Incubate on ice for 30 min.
- Heat shock cells for 60 sec at 42C without shaking.
- Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.
- Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
- In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
- Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.
- Next day, pick and annotate (give each colony a #) 16 colonies onto new LB+Antibiotic plates. Place new plates in 37oC overnight.
- Verify all 16 colonies via PCR with Taq DNA polymerase(DO NOT USE PHUSION!!!) using these primers:
- Run 10µL of all 16 PCR with 2ul of SybrGreen DMSO products onto 1% Agarose gel (110V for 60mins). Expected size-What is your inserts size????
- PICK BEST TWO MUTANTS and start overnight cultures in 2mL LB + Antibiotic. Leave them to grow in the 37C incubator overnight.
- Next morning; miniprep them (there should be 4 plasmids). Measure their concentrations with the nanodrop.
- sequencing
Component | 50μl reaction |
---|---|
Molecular Grade Water | Calculated. Use to make reaction total volume = 50ul |
Custsmart Buffer(different if using pstI) | 5ul |
DNA | 1ug |
Enzyme 1 | .5ul |
Enzyme 2 | 0.5ul |
Component | 20μl reaction |
---|---|
Molecular Grade Water | Calculated. Use to make reaction total volume = 20ul |
T4 DNA Ligase Buffer | 2ul |
Insert DNA | |
Backbone DNA | |
T4 DNA Ligase | 1ul |
Primer A:_________ Tm=__C
Primer B:_________ Tm=__C
Component | 50μl reaction |
---|---|
Molecular grade H2O | Added first. 38.5ul |
10x Standard Taq Buffer | 5μL |
10 mM dNTPs | 1.0μL |
Primer A (10μM) | 2.5μL |
Primer B (10μM) | 2.5μL |
Colony | 1 |
Taq DNA polymerase | Added last. 0.5μL |
Use the following thermalcycler protocol:
Cycle Step | Temp(C) | Time | Cycles |
---|---|---|---|
Initial denaturation | 95 | 5 min | 1 |
Denaturation | 95 | 30s | 35 |
Annealing | LowerTm+1 | 30s | 35 |
Extension | 68 | 41min/kb | 35 |
Final Extension | 68 | 5min | 1 |
Hold | 4 | Hold | 1 |
Gel Electrophoresis
Materials
- Agarose
- 1X TAE Buffer
- Parafilm
- Electrophoresis chamber and power source
- SYBR Green
- Loading Dye
- DNA Ladder
- PCR Samples
Procedure
- Using a balance, mass out 0.5 gram of agarose (for 1% gel, for 2% mass out 1.0 gram). Mix this with 50 mL of TAE 1X Buffer.
- Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).
- Pour the mixture into a gel plate and insert a correctly-sized comb (8, 10, etc. lanes). Allow to cool and solidify on the benchtop.
- Mix your samples on a piece of parafilm. Use 10 μL of the appropriate DNA Ladder (1 kb, 100 bp, 2 log, etc.) combined with 1 μL SYBR Green/DMSO in the first lane. For all samples, mix 10 μl of sample and 2 μL of SYBR Green/Dye Mixture.
- Place gel in tray into the gel electrophoresis apparatus - wells should be close to the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill - there is a guide sticker on the apparatus which marks the max fill line.
- Load 10 μL of each sample into the appropriate wells.
- Connect wiring and run gel electrophoresis at 110 V for 1 hour.
- Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.
Experiments
Growth on Fatty Acids
Materials
Material | Amount |
---|---|
Something | ul |
Something | ul |
Something | ul |
Procedure
- First
- Second
- Third
Terpenoid Growth Curve
Materials
- First
- Second
- Third
Procedure
- First
- Second
- Third
Terpenoid Extraction and Detection
Materials
- First
- Second
- Third
Procedure
- First
- Second
- Third
KillerRed Kill Curve
Materials
- First
- Second
- Third
Procedure
- First
- Second
- Third