1. Introduction
1. Introduction
Candidate genes for detection of SARS-CoV-2 include ORF/RdRp N, E, S gene, and the like. We would like to study RT-PCR (reverse transcription PCR) techniques using N gene as a target.
The reason for RT-PCR is that the genome of SARS-CoV-2 virus is RNA, not DNA.
Since a single strand of RNA is more unstable than a double strand of DNA and RNA cannot be amplified, a process of reverse transcription into stable DNA using un enzyme called RNA-dependent DNA polymerase is required
DNA made through reverse transcription is called cDNA (complementary DNA)
2. Materials and equipment
2. Materials and equipment
- 2x reaction mix [Hot-Taq] (dNTP mix, and include MgCl2)
- Enzyme Mix [Hot-Taq]
- Forward Primer (TGGACCCCAAAATCAGCG:18mer)
- Reverse Primer (GCCTTGTCCTCGAGGGAAT:19mer)
- Template (RNA)
: N gene Positive Control (New England BioLabs. England )
- D.W
- 100bp DNA Ladder
- Conventional PCR used with DuxCycler (BIOMEDUX. Co. Ltd. South Korea)
- Electrophoresis (BIO-RAD Laboratories, Inc., USA)
- Gel-tray (BIONEER Cooperation, South Korea)
- Gel image processing device; DuxGelDoc (BIOMEDUX. Co. Ltd. South Korea)
3. Procedure
3. Procedure
1) Prepare PCR Mixture using experimental ingredients.
Mix 0.2mL PCR tube according to the amount of the table below
| Positive control | Negative control |
|---|
| 2x reaction mix [Hot-Taq] | 10 uL | 10 uL |
| Enzyme Mix [Hot-Taq] | 1 uL | 1 uL |
| Forward Primer | 1 uL | 1 uL |
| Reverse Primer | 1 uL | 1 uL |
| Template | 3 uL | - |
| D.W | 4 uL | 7 uL |
2) The PCR conditions in the table below were set on the device and PCR was performed (running time: about 1.5 hours).
| Processing | Temperature | Time (min.) | Cycles |
|---|
| cDNA synthesis | 50 ℃ | 15:00 | 1 |
| Pre-Denaturation | 95 ℃ | 05:00 | 1 |
| Denaturation | 95 ℃ | 00:20 | 35 |
| Annealing | 50 ℃ | 00:40 |
| Extension | 72 ℃ | 00:30 |
| Final Extension | 72 ℃ | 05:00 | 1 |
| End | 4 ℃* | ∞ |
|
*This is the temperature at which products can be kept stable without denaturation after PCR
3) When PCR is over, the tube is removed from the device and the PCR amplification product is checked using an electrophoresis device.
4. Results
4. Results
| Lane |
|
|---|
| M | 100bp DNA Ladder |
| 1 | PC (1X103 copy/ul) product size : 202bp |
| 2 | NC(negative control) |
*Using the 2% Agarose gel.
The image of the experimental results (DuxGeldoc; BIOMEDUX. Co. Ltd., South Korea)
Results and Interpretation Check whether a 202bp band is formed on the PC(Positive Control). If there is no band, it may be suspected as an error during the PCR process and then, retest should be conducted. And also any band should not be formed in NC (Negative Control). If the band is confirmed on NC, the contamination of the reagents used in the PCR process may be suspected, and in this case, the “re-test” shall be conducted after changing all reagents used in PCR. |
5. Precautions
5. Precautions
-The experimental materials are stored at -20℃ freezer according to the specified storage conditions
-Melt all reagents before starting the experiment
-Never use products that are past the expiration date
-Be careful of contamination during the testing.
#Reverse transcription PCR #RT-PCR #reverse transcription reaction #principle of RT-PCR #Conventional PCR #SARS-CoV-2 #N gene #Electrophoresis
* This RT PCR experiment with the SARS-CoV-2 genomes used educational PCR kit(DuxEdu Kit) of BIOMEDUX Co. Ltd.
Contents
1.Introduction
2.Materials and equipment
3. Procedure
4. Results
5. Precautions
1. Introduction
1. Introduction
Candidate genes for detection of SARS-CoV-2 include ORF/RdRp N, E, S gene, and the like. We would like to study RT-PCR (reverse transcription PCR) techniques using N gene as a target.
The reason for RT-PCR is that the genome of SARS-CoV-2 virus is RNA, not DNA.
Since a single strand of RNA is more unstable than a double strand of DNA and RNA cannot be amplified, a process of reverse transcription into stable DNA using un enzyme called RNA-dependent DNA polymerase is required
DNA made through reverse transcription is called cDNA (complementary DNA)
2. Materials and equipment
2. Materials and equipment
- 2x reaction mix [Hot-Taq] (dNTP mix, and include MgCl2)
- Enzyme Mix [Hot-Taq]
- Forward Primer (TGGACCCCAAAATCAGCG:18mer)
- Reverse Primer (GCCTTGTCCTCGAGGGAAT:19mer)
- Template (RNA)
: N gene Positive Control (New England BioLabs. England )
- D.W
- 100bp DNA Ladder
- Conventional PCR used with DuxCycler (BIOMEDUX. Co. Ltd. South Korea)
- Electrophoresis (BIO-RAD Laboratories, Inc., USA)
- Gel-tray (BIONEER Cooperation, South Korea)
- Gel image processing device; DuxGelDoc (BIOMEDUX. Co. Ltd. South Korea)
3. Procedure
3. Procedure
1) Prepare PCR Mixture using experimental ingredients.
Mix 0.2mL PCR tube according to the amount of the table below
2) The PCR conditions in the table below were set on the device and PCR was performed (running time: about 1.5 hours).
*This is the temperature at which products can be kept stable without denaturation after PCR
3) When PCR is over, the tube is removed from the device and the PCR amplification product is checked using an electrophoresis device.
4. Results
4. Results
*Using the 2% Agarose gel.
The image of the experimental results (DuxGeldoc; BIOMEDUX. Co. Ltd., South Korea)
Check whether a 202bp band is formed on the PC(Positive Control). If there is no band, it may be suspected as an error during the PCR process and then, retest should be conducted.
And also any band should not be formed in NC (Negative Control). If the band is confirmed on NC, the contamination of the reagents used in the PCR process may be suspected, and in this case, the “re-test” shall be conducted after changing all reagents used in PCR.
5. Precautions
5. Precautions
-The experimental materials are stored at -20℃ freezer according to the specified storage conditions
-Melt all reagents before starting the experiment
-Never use products that are past the expiration date
-Be careful of contamination during the testing.
#Reverse transcription PCR #RT-PCR #reverse transcription reaction #principle of RT-PCR #Conventional PCR #SARS-CoV-2 #N gene #Electrophoresis
* This RT PCR experiment with the SARS-CoV-2 genomes used educational PCR kit(DuxEdu Kit) of BIOMEDUX Co. Ltd.