1. Introduction
1. Introduction
It is polymerase chain reaction used to detect tuberculosis bacteria.
Using a universal two-part Primer set of Mycobacterium tuberculosis (MTB) total genome, polymerase chain reaction(PCR) is generated and specific parts are amplified to determine whether tuberculosis is amplified.
2. Materials and equipment
2. Materials and equipment
PCR PreMix, Primer set, MTB DNA, Agaroge gel, TAE or TBE buffer, loading dye, DNA ladder (100bp)
| Forward primer 1 | CCT GCG AGC GTA GGC GTC GG |
| Reverse primer 1 | CTC GTC CAG CGC CGC TTC GG |
| Forward primer 2 | TCC GCT GCC AGT CGT CTT CC |
| Reverse primer 2 | GTC CTC GCG AGT CTA GGC CA |
Conventional PCR (ex. DuxCycler-Biomedux, Ltd. South Korea)
DNA Electrophoresis (ex. DuxGeldoc- Biomedux, Ltd. South Korea)
3. Procedure
3. Procedure
1) The composition of the reaction tube for PCR is as follows. (for one sample)
| Reagent added | Amount of each tube |
|---|
| Template DNA | 3 uL |
| PCR PreMix | 10 uL |
| primer set | 2 uL |
| D.W. | 5 uL |
| Total | 20 uL |
2) when testing the above premix, primer sets, D.W., mix the number of samples by one plus tube and put 17uL into the each PCR tube
3) The template DNA 3uL extracted from the clinical sample is added into the above tube.
(Positive DNA and negative DNA were added to the positive and negative control tubes, respectively.)
4) after spin down the test tubes containing each mixture for 30 seconds, put them in PCR instrument and react as follows
| Processing | Temperature | Time (min.) | Cycles |
|---|
| Pre-Denaturation | 94℃ | 05:00 | 1 |
| Denaturation | 94℃ | 00:30 | 40 |
| Annealing / Extension | 65℃ | 01:00 |
| Final-Extension | 72℃ | 05:00 | 1 |
| End | 4℃ |
|
|
4. Results
4. Results
| Negative | Positive | Positive | Positive |
5. Precautions
5. Precautions
1) Pay attention to pipetting so that DNA or PCR reagents do not contaminated.
2) Be careful not to make mistake in temperature and /or reagent amount during the experimental method.
#Tuberculosis #MTB #DNA # Universal primer #PCR
Contents
1.Introduction
2.Materials and equipment
3. Procedure
4. Results
5. Precautions
1. Introduction
1. Introduction
It is polymerase chain reaction used to detect tuberculosis bacteria.
Using a universal two-part Primer set of Mycobacterium tuberculosis (MTB) total genome, polymerase chain reaction(PCR) is generated and specific parts are amplified to determine whether tuberculosis is amplified.
2. Materials and equipment
2. Materials and equipment
PCR PreMix, Primer set, MTB DNA, Agaroge 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) The composition of the reaction tube for PCR is as follows. (for one sample)
2) when testing the above premix, primer sets, D.W., mix the number of samples by one plus tube and put 17uL into the each PCR tube
3) The template DNA 3uL extracted from the clinical sample is added into the above tube.
(Positive DNA and negative DNA were added to the positive and negative control tubes, respectively.)
4) after spin down the test tubes containing each mixture for 30 seconds, put them in PCR instrument and react as follows
4. Results
4. Results
5. Precautions
5. Precautions
1) Pay attention to pipetting so that DNA or PCR reagents do not contaminated.
2) Be careful not to make mistake in temperature and /or reagent amount during the experimental method.
#Tuberculosis #MTB #DNA # Universal primer #PCR