In our laboratory, we are doing hetero x hetero. Breeding to make knock mouse
Genomic readings are being conducted to test the tails of pups that are about 3 weeks old, classified by genotype (Hetero, wild type, knock out).
2. Materials and equipment
2. Materials and equipment
1.Electropheresis device (Mupid-exU (Takara Korea Biomedical Inc, Takara Japan)
3.0.5x TAE buffer
4.The 3rd Distilled Water
5.Redsafe (iNtRON BIOTECHNOLOGY. Ltd. Korea) = It’s harmless to the human body as a substitute for EtBr.
6.PCR master mix (Takara, Japan)
7.primer (forward, reverse)
12. isopropanol for molecular experiments.
13. ethanol for molecular experiments.
- DNA PREP
1.Cut the tip of the mouse’s tail by 0.5cm put it in the Eppendorf tube
2. Add 750 uL of NTES buffer per tube and 7.5 uL of protein (Proteinase K has keep in freezer. Because of high viscosity, don’t soak the tip and use it from the surface. Otherwise, you ‘ll lose a lot of amount.
3.Vortex and melt it in 56⁰C water bath
4.Visually check if the tail is completely melted and vortex it (there is nothing clumped up and when the hair is scattered, it is completely melted)
5.Since SDS is present in the NTES buffer, centrifugation is performed at 13,200rpm, for 10 minute, at room temperature (there is a possibility that crystals will occur when the temperature is low).
6. Add 500uL isopropanol to the new tube and mix the supernatant 500uL gently (No. pipetting, no vortex).
7.Centriguge in12000rpm, for 10 minute, at Room Temperature.
8.Throw away 500uL of supernatant and add 70% ethanol (for molecular experiment)
9.Centriguge at12,000rpm, for 10 minutes, at Room Temperature.
10.throw away of supernatant and suction with air dry until completely with dry-up (When you don’t do suction turn the tube upside down and dry it).
11.Measurethe HPLC DW by size of pellet, add 100uL ~ 400uL and melt it at least for 3 hours at 4⁰C.
12.Gently pipetting so as not to touch the pellet and centrifuging in 12,000 rpm, for 10 minutes, at 4⁰C
13.Make a PCR mixture, and take an aliquot, 9uL per tube.
14.After pouring 1uL of supernatant, mix it with mixture, and spin down to start PCR.
15.pure agarose powder 1g + 0.5x TAX buffer 100ml mix and microwave until clear (In general 2 minutes)
16.Take it out, mix it in the middle time, then, heat it for another 20 seconds and cool it down.
17. When the gel becomes lukewarm, mix Redsafe 5uL and pour it into the gel mold to harden it for 30~40 minutes.
18.When it‘s hard and firm, start electrophoresis.
19.detect it in UV Detector or with Geldoc.
- Results Image
1.Never vortex in the process of DNA Prep. process.
2.If you make a gel and leave it in the air for too long, it dries, so pay attention to time and electrophoresis.
3In the case of Redsafe, it is not more stable then Et Br, so only 70% of the existing usage is added until gel that has passed more than a day is melted again and used.
4.DNA is dissolved in water, so if left in the refrigerator for a long time, it can be a degradation, so it is frozen.