Standard Agarose Gel Protocol
The kit incorporating four sets of primers allows for the identification of Oil Palm DNA samples as one of six possible genotypes displayed below by visually observing the presence or absence of a PCR amplification product from each primer set in a standard SYBR safe-stained agarose gel electrophoresis.
Below is an example of a standard agarose electrophoresis of PCR products from 3 control DNA genotypes using the 4 different sets of primers included in the SureSawit SHELL Gel Kit. 20 μL PCR product was loaded on a 1% agarose 1X TAE gel and separated.
| Reagents supplied in the kit | |
|---|---|
| Master Mix | 2X master mix containing PCR buffer, dNTPs, and Taq polymerase |
| Primer set 1 | Primer 1 |
| Primer set 2 | Primer 2 |
| Primer set 3 | Primer 3 |
| Primer set 4 | Primer 4 |
| Control 1 | DD - dura Control DNA 1 |
| Control 2 | MP - MPOB pisifera Control DNA 2 |
| Control 3 | AP - AVROS pisifera Control DNA 3 |
| Reagents supplied by the customer | |
|---|---|
| SYBR® Safe DNA Gel Stain | Life Tech Cat # S33102 |
| SYBR® Green I Nucleic Acid Gel Stain | Life Tech Cat # S-7567 |
| Sterile dH2O | |
| 10X Gel Loading Dye |
| Setting up PCR Reaction Note: Each 20 μL PCR reaction will contain DNA, primer, Master Mix (MM) and water. MM and primers are combined in 4 primer cocktail mixes. | |
|---|---|
| Volume | |
| DNA (10ng) Primer Cocktail Mix | 2 μL |
| MM | 10 μ |
| primer | 2 μL |
| dH2O | 6 μL |
| Total: | 20 μL |
- Prepare primer cocktail mixes (1-4) at a volume with overage sufficient for the number of samples plus 3 positive and 1 negative controls. Assemble mixes on ice.
- Aliquot 2 μL DNA to each labelled tube or well of a PCR plate; repeat/multichannel pipettor may be used, however tips should be disposed between samples.
- Add 18 μL of primer cocktail mix to each tube/well and mix by pipetting 3X; multichannel pipette may be used, however tips should be disposed between additions. Assembling the reactions on ice is recommended if working in a warm laboratory environment (>23°C).
- Seal plate and run PCR with the program below.
- Step 1: 94°C 2 min
- Step 2: 94°C 30 sec
- Step 3: 58°C 1 min
- Step 4: 72°C 1 min
- Step 5: Go to step 2 for 34 cycles
- Step 6: 72°C 5 min
- Step 7: 4°C on hold
- After PCR, add 1/10 volume of 10X loading buffer before loading on an agarose gel.
- Load 20 μL PCR product onto a 1X TAE 1% agarose gel containing 1X SYBR Safe DNA Gel Stain (Life Technologies, Cat. # S33102).
- Electrophorese for 60 min at 110V.
- Visualize PCR bands using a blue-light transilluminator and yellow/orange filter or see SYBR Safe manual for recommendations.
Mid-throughput Protocol | SYBR® Green dye detection protocol
