Medino® Mouse Tissue Direct PCR Kit Plus

 

Cat.No. LM201009-2
Size20T/50T/200T/500T
Storage and shipping1. Component A: The product should be stored at 2-8°C for one year. For multiple use for a long time, please avoid cross-contamination.

2. Component B/C: The product should be stored at -20 ℃ for one year. Please avoid repeated freeze-thaw.

3. The product is shipped with dryice.

Product description

This kit can directly and quickly conduct PCR amplification of mouse tissue (such as mouse tail, mouse ear, mouse toe, muscle, etc.), and has strong sample compatibility. Equipped with a powerful lysis buffer, this kit can rapidly lyse samples and release genomic DNA. The lysate can be directly added to the PCR reaction system without purification, and the operation is convenient. 2-5 mm2 mouse ear or liver, 1-5 mm mouse tail, 1-2 toes can be used for experiments.

The 2 × Mouse Direct PCR Mix provided by this kit is a hot-start PCR reaction solution with a 2-fold concentration. It contains all the components used for PCR amplification except the template and primers, which greatly simplifies the operation process and reduces the chance of contamination.

The kit can be used for transgene identification, mouse genotyping, etc.

 

Components

CompontNo.NameFormat
20T50T200T500T
ALysis buffera)5 mL12 mL4×12 mL10×12 mL
BProteinase K(20mg/mL)250μL625μL2× 1.25 mL5× 1.25 mL
C2×Medino® Tissue Direct PCR Mixb)500μL1.25mL5×1 mL10×1.25 mL
  1. a) Lysis buffer containing strong protein denaturants, please wear gloves.
  2. b) 2× Medino® Tissue Direct PCR Mix: Contains hot-start Taq DNA polymerase, dNTP mix, MgCl2, reaction buffer, PCR reaction enhancer, optimizer, stabilizer, electrophoresis.

 

Operate

  1. Sample genomic DNA release

1.1 Cut 2-5 mm2 mouse ear or liver tissue, or 1-5 mm mouse tail, or 1-2 toes, and put them in a 1.5 mL centrifuge tube.

1.2 Add 200 μL Lysis solution and 10 μL Proteinase K to the above centrifuge tube, vortex gently so that the sample is completely infiltrated by the lysate, and centrifuge briefly.

1.3 Incubate at 70°C for 10 min in a constant temperature incubator

1.4 Incubate at 95°C for 5 min Inactivate Proteinase K.

1.5 Vortex and mix the lysate thoroughly, then centrifuge at 12000 rpm (13400× g) for 10 min

1.6 Transfer the supernatant to a new centrifuge tube and store at -20°C for no more than 48 hours or directly use the supernatant for subsequent PCR amplification.

[Note]:

  1. The tissue should be chopped as little as possible to allow the lysis reaction to proceed more smoothly.
  2. Incubate at 70°C, usually 10 min to meet most PCR needs. If a larger amount of DNA is required or the sample is difficult to lyse, the time can be extended to 20- 30 min. The tissue mass does not need to be completely lysed. The residual fraction can be removed in a subsequent centrifugation step.
  1. PCR reaction System
ComponentsVolume μLFinal concentration
2 × Medino® Tissue Direct PCR Mix251 ×
Forward Primer (10 μmol/L)20.4 μmol/L
Reverse Primer (10 μmol/L)20.4 μmol/L
Lysate product (DNA template)1-5
ddH20Up to 50

[Note]: All components should be thoroughly mixed before use.

2.1 Template usage: 2 μL supernatant is recommended as template for 50 μL system.  If the amount of product is small, the amount of template input can be appropriately increased.

2.2 Primer final concentration: it is recommended to use 0.2-0.4 μmol/L of primer final concentration to get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.1-0.5 μmol/L.

2.3 Reaction system: 50 μL is recommended, and the volume of the system can also be adjusted according to usage habits.

2.4 System preparation: Prepare the PCR reaction system, place it on a vortexer, vortex and mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

 

  1. Reaction program
Cycle stepTemp.TimeCycles
Initial denaturation95 °C5 min1
Denaturation95 °C15 sec35
Annealing60 °C15 sec
Extension72 °C3-10 sec/kb
Final extension72 °C5 min1

[Note]:

3.1 Annealing temperature: please refer to the theoretical Tm value of the primer. The annealing temperature can be set 2-5℃ lower than the theoretical value of the primer.

3.2 Extension time: 1-4kb, please set as 3-10 sec/kb. Above 4kb,  please set as 20 sec/kb.

3.3 Number of amplification cycles: 35 cycles can amplify enough products.

3.4 Sample loading by electrophoresis: take 5-7 μL of amplification product and load it.

 

  1. Control response

In the analysis of PCR results, whether positive or negative, the reliability of the results cannot be determined without a control reaction. In order to facilitate the analysis of subsequent experimental results, it is recommended to set positive and negative PCR control reactions during PCR in order to exclude false positive or false negative interference.

Notes

  1. To avoid sample to sample cross contamination, the edge of the sampling equipment or the site in direct contact with the sample was immersed in 2% Sodium hypochlorite solution or 75% alcohol after each sampling session, washed repeatedly several times, and then blotted with clean paper towels to dry the residual liquid before use. For the convenience of the test, multiple sampling equipment can also be prepared and cleaned uniformly after use to ensure that each individual sample uses a non-polluting sampling equipment.
  2. It is recommended to use freshly collected animal tissue. If it is a long-term frozen tissue, repeated freezing and thawing should be avoided as much as possible, otherwise the template will be degraded and the PCR efficiency will be affected.
  3. It is recommended to amplify the fragment length within 6 kb for the best amplification efficiency.
  4. For your safety and health, please wear lab coats and disposable gloves for operation.
  5. This product is for research use ONLY!
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