- Perform a colony PCR
- Run an agarose gel on PCR products
Colony PCR (For 16S rRNA Sequence Analysis)
Polymerase chain reaction (PCR) is molecular technique used to amplify specific regions of DNA for applications such as sequencing and genetic analysis. Typically, there is a limited amount of DNA in the sample to study and amplification is required. PCR is carried out in a test tube with the DNA template, primers specific for the region that is desired, DNA polymerase, and reagents that stabilize the reaction. Once the reaction is put together, it will go into a thermocycler (PCR machine) that will create the conditions for DNA replication to occur. Each round of PCR requires three steps, denaturation, annealing, and elongation, each of which doubles the amount of DNA template present in the reaction. By repeating this process multiple times, usually 30, this will amplify the DNA exponentially.
PCR bead method
· 27F primer (20uM stock)
· 1492R primer (20uM stock)
· GE Illustra PuReTaq Ready to go PCR bead and tube
· Sterile nuclease-free deionized water (molecular grade)
· T-Streak plate with bacterial isolate
· Micropipettors and tips (P10, P100)
Adapted from “GE Illustra PuRe Taq Ready to go PCR beads” guide
- Obtain PCR bead tubes, which contain Taq polymerase (heat resistant enzyme) and other necessary reagents. Using a sharpie, label the top of the tubes with PCR reaction number assigned in class. Make sure not to accidentally rub this off when handling the tube and double check when you put the tube into the PCR machine that your labeling is still visible.
- Add 25 μL of Master mix (contains molecular grade water + 16S rRNA primers) into the PCR bead tube. The bead will start to dissolve and slightly effervesce.
- As you dispense the Master mix, insert the micropipette tip into the mix so that you actually see the small volume go directly into the mix.
- Using a micropipette tip, carefully touch the colony on the streak plate. A small, visible dab of cells that barely fill the very end of the pipette tip will provide enough DNA template for the reaction.
- Dip pipette tip into reaction mix and gently swirl for 5-10 seconds to dislodge cells. Cap the tubes. Avoid forming bubbles.
- Transfer tubes to thermal cycler.
- Select appropriate program† to start cycling (about 2 hours).
- Once cycling is complete, remove tubes and incubate on ice. Follow your instructor’s instructions about storage, and follow up protocols to quality test the PCR products and prepare them for sequencing.
***Protocol adapted from “puRe Taq Ready-To-Go PCR Beads” guide*
16S rRNA Primers:
Forward Primer (27F)
5’ – AGA GTT TGA TCC TGG CTC AG – 3’
Reverse Primer (1492R)
5’ – ACG GCT ACC TTG TTA CGA CTT – 3’
PCR Cycle Protocol:
1. 94oC for 10 min
2. 94oC 30 sec – Denaturation step
3. 58oC 30 sec - Annealing step
4. 72oC 1 min 50 sec (1 min per kb of DNA template) – Elongation step
5. Repeat Steps 2-4 30X
6. 72oC for 10min – Final extension step
Agarose Gel Electrophoresis
For visualizing and analysis, we will have to "run" the PCR products out on an agarose gel. Invitrogen’s E-gel system will be used. This system is a complete buffer-less system for agarose gel electrophoresis. There is a pre-cast agarose gel (E-gel) that is a self-contained gel that includes electrodes packaged inside a dry, disposable, UV-transparent cassette. The gel contains either Sybr-safe or ethidium bromide for visualization of DNA. The E-gel runs in a single device that is both a base and a power supply, called the E-gel Powerbase.
Protocol and images below is adapted from Invitrogen’s E-gel Technical Guide.
· DNA sample (from PCR reaction)
· 1KB Molecular weight markers
· Loading dye Mix
· Run gels stored at room temperature
· Keep samples uniform and load deionized water into empty wells
· Load gel within 15 mins of opening the pouch
· E-gel can only be used once
Sample preparation and Loading gel:
Prepare your DNA samples by adding deionized water to the required amount of DNA to bring the total sample volume to 20ul.
1. The Lab Instructor will add the 1Kb Ladder to the gel.
2. Add 4ul of PCR reaction to new microcentrifuge tube.
3. Add 16ul of Loading dye Mix to this microcentrifuge tube.
4. Once you set up the E-gel powerbase (below), load the entire 20ul volume to the correct gel well. Make sure to note which gel well you loaded your sample into.
Setting up the E-gel Powerbase:
1. Plug the Powerbase into an electrical outlet using the adaptor plug.
2. Open the package containing the gel and insert the gel (with the come in place) into the apparatus right edge first. Press firmly at the top and bottom to seat the gel in the base. You should hear a snap when it is in place. The Invitrogen logo should be located at the bottom of the base, close to the positive pole. See diagram below. A steady, red light, indicates the E-gel is correctly inserted (Ready Mode).
The PCR clean-up process is performed using a commercial product. Depending on the availability of the different commercial kits, your TA will determine and provide the kit to use in lab. Directions will be provided with the kit.