1. Introduction
1. Introduction
An experiment to see whether the amplification results are affected by the storage period or not.
2. Materials and equipment
2. Materials and equipment
TaqMan Gene expression master mix, Primer set (Forward, Reverse, *Probe), Chlamydia trachomatis DNA, Nuclease free water (Qiagen)
- Sequence of Primer, Probe
※ The primer and probe sequence are the same as the #3 NAAT for CT
Real-time PCR (LightCycler 480 – Roche Ltd. Switzerland)
3. Procedure
3. Procedure
1) Prepare Chlamydia trachomatis DNA(10^5 copy)by diluting 10 to 100 times.
2) A sample prepared according to the following PCR sample reagent composition is injected into the 8-strip tube.
| Reagents | Samples | Negative control |
|---|
| Master mix | 15 μL | 15 μL |
| Primer set | 7.5 μL | 7.5 μL |
| CT DNA | 4.5 μL (by conc.) | - |
| Nuclease Free water | 3 μL | 7.5 μL |
| Total | 30 μL | 30 μL |
3) Set the device as below according to as PCR protocol
| Steps | Temperature | Time | Cycles |
|---|
| Initial denaturation | 50℃ | 02:00 | 1 |
| Denaturation | 95℃ | 10:00 | 1 |
| Primers annealing | 95℃ | 00:15 | 40 |
| Elongation | 60℃ | 01:00 |
4) Check the results after the PCR is over.
4. Results
4. Results
- Results Image
<Roche - LightCycler 480 Results>
1) What is marked as new in the sample is a reagent that was subdivided immediately after warehousing and diluted.
2) what is marked as old in the sample is a reagent that has been subdivided after warehousing and stored in the freezer at least more than one month or diluted conditions for a long time.
- interpretation of results
1. Even at the same concentration, it can be seen that the CT value of the OLD sample rises late compared to the NEW sample.
2. When the concentration is low, it can be seen that the CT value rises later.
3. It is considered very important to minimize the freezing and thawing processes by subdividing as aliquot from high concentration of the reagent (for minimizing contamination and deterioration).
5. Precautions
5. Precautions
1) Be careful, do not expose too much to the light.
2) Whenever diluting DNA, be careful to pipette the exact amount.
3) Be careful not to create bubbles in mixed samples
4) Be attention, do not get contaminated every steps during test.
The original author's post (korean) ↗
Contents
1.Introduction
2.Materials and equipment
3. Procedure
4. Results
5. Precautions
1. Introduction
1. Introduction
An experiment to see whether the amplification results are affected by the storage period or not.
2. Materials and equipment
2. Materials and equipment
TaqMan Gene expression master mix, Primer set (Forward, Reverse, *Probe), Chlamydia trachomatis DNA, Nuclease free water (Qiagen)
※ The primer and probe sequence are the same as the #3 NAAT for CT
Real-time PCR (LightCycler 480 – Roche Ltd. Switzerland)
3. Procedure
3. Procedure
1) Prepare Chlamydia trachomatis DNA(10^5 copy)by diluting 10 to 100 times.
2) A sample prepared according to the following PCR sample reagent composition is injected into the 8-strip tube.
3) Set the device as below according to as PCR protocol
4) Check the results after the PCR is over.
4. Results
4. Results
- Results Image
<Roche - LightCycler 480 Results>
1) What is marked as new in the sample is a reagent that was subdivided immediately after warehousing and diluted.
2) what is marked as old in the sample is a reagent that has been subdivided after warehousing and stored in the freezer at least more than one month or diluted conditions for a long time.
- interpretation of results
1. Even at the same concentration, it can be seen that the CT value of the OLD sample rises late compared to the NEW sample.
2. When the concentration is low, it can be seen that the CT value rises later.
3. It is considered very important to minimize the freezing and thawing processes by subdividing as aliquot from high concentration of the reagent (for minimizing contamination and deterioration).
5. Precautions
5. Precautions
1) Be careful, do not expose too much to the light.
2) Whenever diluting DNA, be careful to pipette the exact amount.
3) Be careful not to create bubbles in mixed samples
4) Be attention, do not get contaminated every steps during test.
The original author's post (korean) ↗