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Team:UCSF/Protocols
From 2010.igem.org
AarI Digest Protocol
1. Label PCR tubes.
2. Add the following reagents into the PCR tubes:
Aar1 Digest Reagents:
5 ug DNA
2.5ul Aar1 Enzyme
0.9ul Aarl oligo
6 ul 10x Aar1 Buffer
x ul dH2O
60 ul total reaction
Note: x ul dH2O may change in volume for different reactions in order to bring total volume up to 60 ul.
3. Briefly vortex and spin down the reaction.
4. Incubate reaction at 37C for 3 hours.
Note: PCR program can be set to 37C for 3 hours and cooled down to 4C indefinitely.
PCR Phusion Protocol
1. Label PCR tubes.
2. Add the following reagents into the PCR tubes (in order):
PCR Reagents
23.7 ul dH2O
10 ul 5x HF Buffer
5 ul forward primer
5 ul reverse primer
5 ul dNTP
0.3 ul template
1 ul Phusion
50 ul total
3. Vortex and spin down the reaction.
4. Set up PCR program.
| Cycle Step | Temperature | Time | # Cycles | Initial Denature | 98C | 3min | 1 |
| Denature Annealing Extension | 98C 55C 72C | 10s 30s 30s | 30 | ||||
| Final Extension | 72C | 5min | 1 | ||||
| Hold | 4C |
Note: Times may differ for different reactions based on the size of the desired PCR product. Annealing temperature may also differ depending on primer properties.
Ligation Protocol
Ligation Reagents:
50 ng vector
DNA insert(s)
Buffer
1 ul Ligase
Ligation Reaction:
1. Calculate amount of DNA insert and vector needed for reaction.
150 ng / ( # bp in backbone/ # bp in insert) = ng needed
Concentration of insert or vector divided by ng needed = volume of DNA needed
2. Calculate amount of buffer needed depending on concentration for volume needed. A typical ligation reaction volume can be 20 ul.
3. Add reagents together, from smallest volumes to largest. Distilled water can be used to bring the volume up so that the reaction has the proper buffer concentration for the reaction. Ligase or enzymes in general should be added at the end.
4. Vortex or pipet up and down to mix. Spin down afterwards if vortexing.
5. Let reaction sit at room temperature for 5 to 20 minutes depending on ligase used. The ligase may start acting up if left too long.
Gel Electrophoresis Protocol
Gel Electrophoresis Reagents/Materials:
Gel caster
Gel tray
Agar powder
TAE buffer
10, 000X Sybr Safe
50ml conical tube
Gel comb
Microwave
Large flask (microwave safe)
Preparing the agarose solution:
1. Pour in flask the amount of TAE buffer desired. This will be the volume of agarose solution made.
2. Measure out the amount of agar needed for desired concentration. For example to get a 1% agarose gel, you would add 1 gram of agarose powder to 100 ml of TAE buffer. Most concentrations commonly fall between 0.7- 2%. Higher concentrations of agarose are better suited for small fragment separation while lower concentrations are suitable for large fragments.
3. Microwave three minutes, may vary depending on volume in flask. Make sure cap is on loosely.
4. Using hot gloves, swirl bottle gently to ensure complete dissolving of agar. The solution should be clear when agar is completely dissolved. If it is not, microwave for another minute.
5. Place flask of agarose solution in 80C bath to store as to prevent solidifying of agarose solution.
Casting gel:
1. Secure gel tray in caster
2. Pour desired volume of premade agarose solution into 50 ml conical tube. Volume desired will vary depending on gel tray size and desired thickness of gel. Thin gels solidify quicker and can show faint bands clearer, but the wells of thicker gels can hold more DNA.
3. Add Sybr safe to agarose solution. For 10 ml of agarose solution, add 1ul Sybr Safe. The Sybr safe is used to stain and visualize the DNA when viewed under a blue light.
4. Invert tube several times until Sybr safe is equally distributed. Invert gently to avoid creating air bubbles.
5. Pour contents of conical tube into secured gel tray.
6. Place gel comb into notches of gel tray. Check that comb is evenly submerged in agarose. The comb will form wells in the gel to load DNA.
7. Dry at room temperature. The gel will appear opaque when done.
