1. Introduction
1. Introduction
It is a polymerase chain reaction analysis method used to diagnose salmonellosis using a primer set capable of amplifying two major typhoid genes.
2. Materials and equipment
2. Materials and equipment
PCR PreMix, Primer set, Salmonella DNA, Agarose gel, TAE or TBE buffer, loading dye, DNA ladder (100bp)
| Forward primer 1 | GCT TAA TGT CCA AGA TGC CTA CAC |
| Reverse primer 1 | GAG CAA CGC CAG TAC CAT CTG |
| Forward primer 2 | CAC GCA CCA TCA TTT CAC CG |
| Reverse primer 2 | ACT AAC GGC CTA AAC GGG AT |
Conventional PCR (ex.DuxCycler-Biomedux. Ltd., South Korea)
DNA Electrophoresis (ex.DuxGeldoc- Biomedux. Ltd., South Korea)
3. Procedure
3. Procedure
1) Prepare a PCR mixture. The PCR method solution is prepared by mixing each reagent with the composition of the following table based on one sample. At this time, the amount of each reagent used is calculated according to the number of reactions of the sample including the control group (positive control group, negative control group)
| Reagent added | Amount for each tube |
|---|
| PCR Premix | 10 uL |
| Primer mix | 4 uL |
| DW | 3 uL |
| Total | 17 uL |
2) Mix the PCR mixture 5 times, vortex it, and centrifuge it lightly.
3) Pour the PCR mixture into the PCR tube 17 uL on each tube (close the lid immediately after pouring to prevent contamination)
4) Add template DNA 3 uL extracted from clinical samples to the tube.
(The positive control group DNA provided is added to the positive control group, and D.W. is added to the negative group)
| Reagent added | Amount (for each tube) |
|---|
| PCR mixture | 17 uL |
| template DNA | 3 uL |
| Total | 20 uL |
4) each mixed solution tube is centrifuged for 30 seconds and then put into a PCR machine to react as follows.
| Processing | Temperature | Time(min.) | Cycles |
|---|
| UDG treatment | 37℃ | 02:00 | 1 |
| Pre-Denaturation | 95℃ | 15:00 | 1 |
| Denaturation | 95℃ | 00:30 | 40 |
| Annealing | 58℃ | 01:00 |
| Extension | 72℃ | 02:00 |
| Final-Extension | 72℃ | 05:00 | 1 |
| End | 4℃ | ∞ |
|
5) Check the PCR product 5 uL by electrophoresis in 1~2% agarose gel.
4. Results
4. Results
| Lane |
|
|---|
| SM | Size Marker |
| 1 | Positive |
| 2,3,4 | Negative |
-Both bands are determined to be positive when confirmed at 587bp and 484bp.
5. Precautions
5. Precautions
1) Pay attention to pipetting so that DNA or PCR reagents are not contaminated during the testing.
2) Be careful not to make a mistake in temperature and or amount of reagent during the experiment is performed
#Salmonellosis #DNA # UDG system #PCR
Contents
1. Introduction
2. Materials and equipment
3. Procedure
4. Results
5. Precautions
1. Introduction
1. Introduction
It is a polymerase chain reaction analysis method used to diagnose salmonellosis using a primer set capable of amplifying two major typhoid genes.
2. Materials and equipment
2. Materials and equipment
PCR PreMix, Primer set, Salmonella DNA, Agarose gel, TAE or TBE buffer, loading dye, DNA ladder (100bp)
Conventional PCR (ex.DuxCycler-Biomedux. Ltd., South Korea)
DNA Electrophoresis (ex.DuxGeldoc- Biomedux. Ltd., South Korea)
3. Procedure
3. Procedure
1) Prepare a PCR mixture. The PCR method solution is prepared by mixing each reagent with the composition of the following table based on one sample. At this time, the amount of each reagent used is calculated according to the number of reactions of the sample including the control group (positive control group, negative control group)
2) Mix the PCR mixture 5 times, vortex it, and centrifuge it lightly.
3) Pour the PCR mixture into the PCR tube 17 uL on each tube (close the lid immediately after pouring to prevent contamination)
4) Add template DNA 3 uL extracted from clinical samples to the tube.
(The positive control group DNA provided is added to the positive control group, and D.W. is added to the negative group)
4) each mixed solution tube is centrifuged for 30 seconds and then put into a PCR machine to react as follows.
5) Check the PCR product 5 uL by electrophoresis in 1~2% agarose gel.
4. Results
4. Results
-Both bands are determined to be positive when confirmed at 587bp and 484bp.
5. Precautions
5. Precautions
1) Pay attention to pipetting so that DNA or PCR reagents are not contaminated during the testing.
2) Be careful not to make a mistake in temperature and or amount of reagent during the experiment is performed
#Salmonellosis #DNA # UDG system #PCR