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Lab Notebook Overview

Common Protocols We Used

Protocol: PCR

Materials

• Forward & Reverse Primers
• PCR bead in tube (bead contains polymerase and nucleotides)
• 19 µL of deionized water (we used 16 µL by mistake and it still worked)
• 1 µL of template plasmid
• 2.5 µL of 10x forward primer solution
• 2.5 µL of 10x reverse primer solution

Procedure: PCR Primer DNA

Forward and reverse primer DNA are shipped in separate storage tubes. The first step is to make a 100X solution of each in the storage tubes by adding deionized water.

Add the appropriate amount of water and set aside.

Make 10x solution of the primer DNAs

In separate small Eppendorf tubes, mix 10 µL of the primers with 90 µL of deionized water. Close top and vortex each tube. Make sure to label the tubes. Set aside.

PCR Reaction

The objective in this step is to clone the DNA in the template plasmid (GFP in our case).

Step 1: Add deionized water to PCR tube. Flick tube to dissolve PCR bead. (DO NOT VORTEX – amount is too small.)

Step 2: Add template plasmid, forward primer, and reverse primer. Flick tube to mix (DO NOT VORTEX)

Step 3: Place in PCR machine and run through cycle (takes about 90 minutes)

Step 4: Remove & label tube

If all went well, you should have a tube of lots of the template DNA with blunt ends.

*BREAKPOINT* (A breakpoint is a place where you can stop working. Please make sure all of your test tubes are labeled and placed in the freezer in the iGEM box.)

Protocol: PCR Purification

In this next step we need to remove the polymerase from the test tube. A later step in this process will be to convert the blunt ends of the GFP DNA into sticky ends. If the polymerase is not removed then it will be impossible to create the sticky ends since as the sticky ends are created, the polymerase will convert them back to blunt ends. The purification process is done with a kit. In our lab we used the PureLink Quick Gel Extraction and PCR Purification Combo made by Invitrogen (Catalog number: K2200-01)

Step 1: Combine. Add 4 volumes of Binding Buffer (B2) with isopropanol to 1 volume of PCR sample (in our case, 88 µL of B2). Mix well.

Step 2: Load. Pipet the sample into a PureLink Clean-up Spin Column in a Wash Tube. Centrifuge the column at >10,000 x g for 1 minute. Discard the flow through.

Step 3: Wash. Re-insert the column into the Wash Tuben and add 650 µL Wash Buffer (W1) with ethanol. Centrifuge the column at >10,000 x g for 1 minute.

Step 4: Remove ethanol. Discard the flow-through and place the column in the same Wash Tube. Centrifuge the column at maximum speed for 2-3 minutes.

Step 5: Elute. Place the column into a clean 1.7 mL Elution Tube. Add 50 µL Elution Buffer (E1) to the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum for 1 minute.

The elution tube contains the purified PCR product. Make sure to label the tube.

*BREAKPOINT*

Protocol: Run a Gel

Now that we have a purified PCR produce we would like to confirm that we actually have some DNA that appears to be correct. To do this, we will run a gel. GFP contains approximately 783 base pairs so we will run 100 bp ladder to compare the PCR product to.

The first step is to make a gel of the right concentration. Gels concentrations run 0.7% - 2%.

For 500 bp sequences, concentration of > 1% is good. For 783 bp, a 1% gel should work.

Make the gel:

Materials:

• 0.5 g of agarose
• 50 mL of TAE
• 5 µL of Ethidium Bromide (for small gel)
• Small Electrophoresis box

Step 1: Measure .5 g of agarose. Use folding paper (fold in quarters first). Also be sure to take scale after putting on folding paper.

Step 2: In 250 mL Erlenmeyer Flask, mix agarose and 50 mL of TAE. Swirl flask to dissolve most of the agarose. Liquid will be foggy. Cover flask with Saran Wrap and microwave for 2 minutes at high. Liquid will be clear.

Step 3: Add 5 µL of Ethidium Bromide. Swirl.

Step 4. Set up electrophoresis box to make gel. Make sure tray is oriented so that liquid gel will not leak out. Insert well mold. Pour in enough gel mix to fill tray. Let sit for about 15 minutes (will turn foggy when ready) to allow gel to set. Rinse flash in tap water.

Step 5: Add a splash of TAE to lubricate gel. Wiggle out well mold. Gently take out tray and rotate 90 degrees. Make sure that wells are on NEGATIVE side (black lead) or sample will run the wrong direction (DNA is negative and will be drawn to the positive side).

Step 6: Fill gel box with TAE 1% solution. It should just go over the top of the gel.

Step 7: Get DNA sample ready to run gel. Place a small piece of parafilm over a test tube tray and make a small indentation (area will be used to mix DNA and loading dye). Mix 2 µL (we used 9 in error) of DNA sample with 1 µL of loading dye in parafilm indentation. Using pipette, place full mixture into one of the gel wells. In a neighboring well, place in 3 µL of 100 base pair ladder.

Step 8: Run the gel by plugging leads into the transformer. If power is on, you will see small bubbles coming off of wires. Let the gel run until the dye has gone about 1/3 the way across the gel.

Step 9: To see the actual DNA, take the gel tray and view it under ultraviolet light. If everything worked you will see one band in the DNA column and multiple bands in the ladder column. See where the DNA band lines up against the ladder to estimate the number of base pairs and to see if they correspond to the expected amount. Take a picture of the gel.

Materials

• 9.5 µL of deonized water
• 6 µL of GFP DNA
• 1 µL of EcoRI (restriction enzyme)
• 1.5 µL if PstI (restriction enzyme)
• 2 µL of buffer (must match restriction enzymes used – check with lab director)

Procedure: Digest a PCR product

IMPORTANT: Add materials in this order:

• Water
• Buffer
• DNA
• Restriction Enzymes

Flick to mix.

Heat in PCR machine at 37 degrees C for 1 hour

Ice mixture