Build-a-Gene Session 5

COLONY PCR

Because PCA produces many DNA molecules, not all of which are the correct size, we want to make sure that the DNA that we work with from here on contains the full-length emGFP gene. Remember that each bacterial cell originally picked up one DNA molecule. As that cell grew into a colony, all of the cells in that colony contain the same DNA molecule. Other bacterial colonies will contain DNA molecules that may be of a different size or sequence. We can therefore screen the bacterial colonies by colony PCR to determine which ones contain a plasmid insert that is the correct size for the emGFP+promoter.

1. Dispense 50 ul of water per tube into 12 different tubes.
2. Use a sterile toothpick to pick a bacterial colony and resuspend it in a tube with water. Repeat for 11 more colonies.
3. Prepare a master mix for all PCRs by combining all reagents listed below in one tube.

10 uM primer	8.4 ul
10 uM primer	8.4 ul
2.5 mM dNTPs	14.0 ul
Taq+buffer	95.2 ul

4. Add 9 ul of the master mix into 12 different PCR tubes.
5. Add 1 ul of resuspended bacterial cells from a different colony into each PCR tube (IT IS VERY IMPORTANT TO STORE THE REMAINDER!) Start the PCR reactions in the PCR machine.

Reaction Conditions:

95°C, 6 minutes

30 cycles:

       95oC, 30 seconds
       55oC, 30 seconds
       72oC, 1 minute

72oC, 10 minutes

GEL ELECTROPHORESIS

Now, we need to check how well our colony PCR worked by running our PCR products on an agarose gel to verify whether which colonies contain a plasmid carrying the full-length emGFP gene.

Pouring a Gel:

1. Weigh out 0.35 g of agarose on a piece of weigh paper. Transfer to an Erlenmeyer flask. Add 50 ml of 1x TAE.
2. Place the flask in the microwave and heat until the agarose is completely transparent and colorless.
3. Remove the flask of clear agarose and allow it to cool. This will take about 10 min.
4. When the agarose is cool, add 5 ul of gel red to the melted agarose
5. Swirl the agarose to incorporate the gel red and pour the agarose into the gel tray.
6. Allow at least 20 minutes for the gel to solidify. Once solid, carefully remove the comb and place the solidified gel (still on the tray) into the gel box so that the wells are oriented on the same side as the black electrode.
7. Add enough 1x TAE buffer to completely cover the gel by about 1 cm.

Preparing your samples:

1. On a piece of parafilm, spot out 2 ul of 6x DNA loading dye (for each colony PCR reaction.
2. Add 5 ul of water to each spot of dye.
3. Add 5 ul of PCR product to each spot of dye.

Running a Gel:

1. Into the first lane of the gel load 10 ul of the DNA ladder.
2. Then load 10 ul of each of your PCR products (mixed with water and dye).
3. Place gel lid with electrodes on gel box, and set voltage to 100V.
4. Run gel approximately 30 minutes or until the dye is 2/3 of the way down the gel, then take picture.