Team:UCSF/Protocols

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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.

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.

Colony PCR protocol

Colony PCR reagents:

12ul dH20

4 ul 5x gotaq green buffer

2 ul 10mM dntp

1 ul forward primer

1 ul reverse primer

0.1 ul GoTaq Polymerase

1. After adding reagents to PCR tube, pick a colony and touch the bottom of the PCR tube with the pipette.

2. Place PCR tubes into the PCR machine and set the cycling parameters to be optimal for the piece of DNA being copied.

Transformation Protocol

Transformation Reagents

LB Plates

Ice box

Water bath

37C Incubator

Competent cells & Plasmid

Spreading tool (beads)

1. Prewarm LB plates in the 37C incubator

2. Remove desired cells from -80C freezer (or other storage location) and melt over ice for at least 10 minutes. (Make sure to aliquot the correct amount of cells for transformation)

3. Pipette up 5ul of your plasmid DNA and gently pipette into the suspended cells. Do not mix by triterating the solution (pipetting up and down).

4. Let the cells sit on ice for 15-30 minutes.

5. Heat shock your cells for 75 seconds in a 42C water bath.

6. Quickly return your tubes to the ice bucket for 2-3 minutes.

7. When you are ready to plate, remove your prewarmed plate from the incubator. Using sterile technique, carefully pipette your entire tube of cells onto the LB plate.

8. Again using sterile technique, spread the cells across your plate using either sterile beads or another spreading tool.

9. Put the plate in the 37C incubator for 5-15 hours.

Gel Extraction/Purification protocol

Gel Extraction/Purification Materials & Reagents:

Sterile Blade

2ml microcentrifuge tube

1.5ml microcentrifuge tube

QIAGEN Gel Extraction Kit

Centrifuge

Heat Block/Bath

1. Using a sterile blade, carefully excise the desired band from your gel.

2. Transfer the agarose chunk into a new 2ml microcentrifuge tube.

3. Weigh the excised agarose chunk and add 3 times its weight in microliters of QG buffer.

4. Move the tube to a melting block for 10 minutes at 50C or until the agarose is completely melted.

5. Add isopropanol to the tube at a 1:3 ratio with QG, then vortex the solution briefly to help precipitate the DNA.

6. Transfer the solution to a Qiagen gel extraction/PCR purification spin column.

7. Spin for 1 minute at 13,000rpm and dump the supernatant.

8. Add 500μl of QG buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum the flowthrough.

9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum flowthrough.
NOTE: Wash the rim with the buffer.

10. Repeat Step 9

11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum)

12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column.

13. Place tubes in a -20oC freezer box.

Miniprep protocol

Miniprep Materials

Qiagen Miniprep Kit

Centrifuge

1. Spin down bacterial culture at 3500 rpm for 5 minutes

2. Aspirate supernatant. Change tips for every sample.

3. Resuspend in 250μl of P1 Buffer. Vortex until cell pellet is not visible. Transfer into a 1.5 ml tube.

4. Add 250μl of P2 Buffer. Invert 5-6 times. Wait 3-5 minutes.
NOTE: When you open the cap, you should see a string of DNA.

5. Add 350μl of N3 Buffer. Invert 5-6 times or until colorless

6. Centrifuge at 13,000 rpm for 10 minutes. Label blue spin columns. Transfer supernatant into blur spin columns.

7. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant.

8. Add 500μl of PB buffer. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant.

9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum supernatant.
NOTE: Wash the rim with the buffer.

10. Repeat Step 9

11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum)

12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column.

13. Place tubes in a -20oC freezer box.