Animal Tissue Direct PCR Kit-UNG

Composition

Storage

Transportation conditions: Low temperature icebox transport throughout the whole process to ensure the kit is kept below 4℃.

Storage conditions: 2×PCR Mix (UNG plus) should be stored at -20℃, others reagents can be stored at 2-8℃.

Introduction

This kit uses a unique lysis buffer system to quickly release genomic DNA from animal tissue samples for PCR, it is particularly suitable for large-scale genetic testing.

The process of releasing genomic DNA from the lysis buffer is completed within 10-30 min at 65℃. No other processes such as protein and RNA removal are required, and the released trace DNA can be used as a template for PCR.

2×PCR Mix (UNG plus) uses dUTP instead of dTTP on the basis of 2×PCR Mix, and adds UNG (Uracil-N-glycosylase) that can degrade the template containing dUTP at the same time. Before the PCR reaction, the UNG is used to degrade the PCR product containing uracil. The UNG will not have any effect on the template that does not contain uracil, thereby ensuring the specificity and accuracy of amplification and preventing the possibility of PCR products contamination during large-scale gene detection.

Equipment and Reagents to Be Supplied by User

1.       1.5 ml microcentrifuge tubes, Pipettes and pipette tips (DNase-free & RNase-free tips with filter element recommended)

2.       Protective equipment such as disposable latex gloves and paper towels

3.       Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)

4.       Vortexer

Preparation Before Use

1.       Pay attention to the cleaning of experimental equipment and the experimental operation to avoid cross-contamination between samples.

2.       Please try to use freshly collected animal tissue samples for experiments. If the tissue samples are stored for a long time, please avoid repeated freezing and thawing of the samples.

3.       If there is precipitation in Buffer AL, it can be placed at 37℃ until the precipitation disappears, and the solution is shaken before use.

4.       2×PCR Mix (UNG plus) should avoid repeated freezing and thawing, otherwise it will affect the PCR detection.

5.       During electrophoresis detection, do not use Loading Buffer containing SDS, otherwise a large group of tailing bright bands will appear in the swimming lane during electrophoresis. It will affect the experimental results.

Prevent Cross-contamination Between Samples

To avoid cross-contamination between samples, after each sampling, the cutting edge of the sampling equipment or the part directly in contact with the sample needs to be immersed in 2% sodium hypochlorite solution, washed repeatedly for several times and wiped off the residual liquid with clean paper towel and then it can be used again. In order to facilitate the test, multiple sampling equipment can also be prepared and cleaned uniformly after use to ensure that each individual sample uses non-polluting sampling equipment.

Protocol

A.     Sample DNA Release

1.       Add 100 μl of Buffer AL and 4 μl of Proteinase K to a 1.5 ml microcentrifuge tube, vortex gently to mix.

* The mixture of Buffer AL and Proteinase K should not be stored for a longtime, please use it as soon as possible.

2.       Cut 5-10 mg of animal tissue into the above 1.5 ml microcentrifuge tube, and vortex gently to mix.

* Trim animal tissue as much as possible to facilitate the enzymatic hydrolysis reaction.

3.       Incubate at 65℃ for 10-30 min, then treat at 95℃ for 5 min.

* Incubation at 65℃ generally only takes 10 minutes to meet most PCR needs. If the amount of DNA required is large or the sample is difficult to digest, the time can be extended to 30 min. The tissue block does not need to be completely digested, and the residual fraction can be removed in a subsequent centrifugation step.

4.       Centrifuge at 12,000 rpm (~13,400×g) for 5 min.

5.       Transfer the supernatant to a new 1.5 ml microcentrifuge tube and store at 4℃ or -20℃ for later use or directly for PCR amplification.

B.     PCR amplification

1.       Add the corresponding 2×PCR Mix (UNG plus) and specific primers to PCR tube for use.

2.       Take an appropriate amount of the lysis mixture processed in step A and add it to the PCR system prepared above (See Table 1 for system preparation).

* When used for subsequent PCR detection, the optimal amount of template accounts for 1-10% of the PCR system and cannot exceed 20%. For example, in a 50 μl PCR system, 0.5-5 μl of lysate can be added, but not more than 10 μl.

3.       Carry out the PCR according to the optimized PCR conditions (annealing temperature, etc.) (See the following table 2 for the reaction conditions).

* Try to use the optimized conditions for PCR to get better results.

4.       Agarose gel electrophoresis test results.

* It is recommended to use the 6×Loading Buffer delivered with the kit. Do not use Loading Buffer containing SDS for electrophoresis.