genotyping

Summary

Mouse genotyping with mouse tail or toe or tissue

Principle

The mouse genome DNA is obtained by chemical cleavage of the mouse tail or toe or tissues and amplified by PCR, and the mouse genotype is determined by horizontal electrophoresis of the DNA.

Appliance

Identification of mouse genotypes

Operation method

Identification of mouse genotypes

Principle

The mouse genome DNA is obtained by chemical cleavage of the mouse tail or toe or tissues and amplified by PCR, and the mouse genotype is determined by horizontal electrophoresis of the DNA.

Materials and Instruments

Reagents:
NaOH, EDTA, Trizma base
Genotype amplification kit, agarose, TAE solution, nucleic acid dye
Name
Formulation
pH
Liquid A
0.1 g NaOH + 0.058 g EDTA + 100 mL ddH
2
O
Liquid B
0.63 g Trizma base + 60 mL ddH 2 O
2
O
Adjust pH = 8.0
Instruments:
Ophthalmic scissors, forceps, metal bath heater, PCR apparatus
Horizontal electrophoresis equipment (main unit, electrophoresis tank)

Move

1. Cut off an appropriate amount of mouse toe, tail or tissue (at least one toe or 0.5 cm long tail) and place it in a 1.5 mL EP tube. Add 100 μL of liquid A, mix and centrifuge, and cook at 100 ℃ in a metal bath for 1 hour, removing and centrifuging once.

After cooking, let it stand at room temperature, then add 100 μL of liquid B and mix well, then the genotype can be determined immediately, or stored at -20 ℃ for a long time.

3, take the supernatant, according to the gene amplification kit instructions to configure the amplification system, generally 10 μL of Taq enzyme Mix, 2 μL of supernatant, 1 μL of 1x primer in each sample, and finally make up to 20 μL with _ddH2O.

Note that a blank control ( _ddH2O as template) and a wild-type control (wild-type mouse sample as template) were prepared at the same time. The amplification system was mixed and centrifuged, then amplified in the PCR instrument, and the amplified DNA was stored at 4 ℃ for a short period of time.The PCR program settings can be adjusted according to the primer design, and the routine can be considered:

Temperature	Time	Cycle
94 °C	5 min
94 ℃	30 s	35 cycles
94 °C	30 s	35 cycles
55 °C	30 s
72 ℃	30 s
72 ℃	7 min
4 ℃	∞

4, the preparation of agarose gel, according to 60 mL of 1xTAE solution add 0.9 g of agarose proportion of preparation, mixing, microwave oven to medium-high heating for 3 minutes to confirm that the agarose powder is completely dissolved until the liquid is slightly cooled down, add 6 μL of nucleic acid dye, shake and mix well and poured into the gel plate and wait for the gelatinization.

5, the solidification time is about 30 minutes, then pull out the comb and put the gel into the electrophoresis tank, add TAE solution to diffuse over the gel, 10 μL of sample per well, in addition to adding samples, and also add a blank control, a wild-type control and a DNA ladder. the horizontal electrophoresis voltage is set at 120 V, and the time is determined according to the size of the bands, generally 20~30 minutes. At the end of electrophoresis, observe the position of the bands under the gelator to determine the genotype of the mice.

Caveat

1) Ensure clean operation, doping of other substances may lead to the appearance of stray bands;2) ddH2O with freshly sterilized, or use DEPC water.3) When configuring the agarose gel, add the nucleic acid dye when the agarose is completely dissolved and mix it well, then quickly introduce it into the gel-making plate, avoid cooling for too long to avoid flocculent precipitation when cooling down.In addition, if air bubbles appear after pouring into the gel plate, use the tip of the drying gun to pick up the bubbles, and the small bubbles will not affect the results.

Common Problems

(1) Spurious bands: Compare with the blank control, firstly, check whether _ddH2O is the problem; secondly, check whether the annealing temperature is suitable for the amplification of the primer, and generally the annealing temperature is 1~2 ℃ lower than the Tm value of the primer.

(2) No or very weak bands: compare with the wild-type control, firstly, check whether the result is the correct genotype of the mouse; secondly, check whether the amount of DNA is too low due to too little sample cutting or insufficient cleavage.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/