Notch translation experiment

Summary

Source : Molecular Cytogenetics Techniques and Applications

Operation method

basic program

Principle

Notch panning is a method of admixing labeled nucleotides into DNA, which can be either an isolated DNA fragment or a complete clone. The method utilizes the combined action of two enzymes: DNAzyme I opens a nick in the DNA strand to form a 3' end light group, and DNA polymerase I adds a nucleotide to the 3' end hydroxyl group. As DNA polymerization proceeds, the 5'-3' exonuclease activity of DNA polymerase hydrolyzes the nucleotide at the 5' end of the nick. Labeled and unlabeled nucleotides are doped in as the reaction proceeds, and DNA fragments of various sizes are synthesized, but there is no net synthesis of DNA. The resulting double-stranded DNA fragments must be denatured before hybridization.

Materials and Instruments

The sample DNA to be labeled generally contains 1 μg of DNA in 50 μL of the labeling reaction.
Fluorescein-labeled dUTP Working solution concentration of 0.2 mmol L dATP, dCTP, dGTP, dTTP all 0.3 mmol L . Notch panning enzyme mixture 10× notch panning buffer TAE buffer
Water bath PCR instrument

Move

1. Preparation of 0.1 mmol/L dTTP solution: 100 μL of 0.3 mmol/L dTTP was added to 200 μL of nuclease-free water.

2. Preparation of 0.1 mmol/L dNTP mixture: mix 100 μL each of 0.3 mmol/L dATP, 0.3 mmol/L dCTP and 0.3 mmol/L dGTP. The excess nucleotide mixture can be stored at -20°C or -80°C.

3. For each labeling reaction, add 1 μg of DNA, 0.2 mmol/L fluorophore-labeled dUTP 2.5 μL, 0.1 mmol/L dTTP 5 μL, 0.1 mmol/L dNTP mixture 10 μL, 10X notch panning buffer 5 μL, and nuclease-free water to 40 μL to a small centrifuge tube placed on ice

4. Add 10 μL of notch pan enzyme mixture to each small centrifuge tube.

5. Mix well and centrifuge slightly.

6. Incubate at 15℃ for 8~16h.

7. Add 5μL of reaction termination solution.

8. To remove unadulterated nucleotides, add sodium acetate, 125 μL of anhydrous ethanol, and centrifuge at 12,000 r/min for 20 to 30 min. Carefully pour off the supernatant or aspirate it with a micropipette. Add 100uL of 70% ethanol, shake slightly, and centrifuge at 1500g for 5min. carefully pour off the supernatant or aspirate the supernatant with a micropipette.

9... Treat the sample with a vacuum freeze dryer.10~20min. resuspend the precipitate with 10pLTE to give a final concentration of about 100ng/μL. Undoped nucleotides can also be removed with a Sephadex 050 centrifugal column, following the product instructions. After treatment with a centrifugal column, the volume of labeled DNA ranges from 50 to 100 μL, which is then concentrated according to step 8.

10. To determine the size of the labeled DNA fragments, add 0.5 g of agarose to 50 mL of TAE or TBE buffer and carefully heat to boiling using a microwave oven or hot plate. When all of the agarose is dissolved, lower the temperature to 55°C in a water bath.

11. Add 2.5 μL EB to the agarose and mix well, then pour the agarose onto a glue-laying plate with a 12- or 16-well comb.

12. For each labeled DNA sample, mix 2 μL of DNA (~200 ng), 7 μL of water (TAE or TBE), and 1 μL of upsampling buffer.

13. Electrophoresis each labeled sample and molecular mass reference at 70 ~ 100V until the preceding stain migrates about 5 cm in the gel.

14. Observe the DNA on a UV projection reflectometer and take a picture. Most of the DNA fragments should be at 200 ~ 600bp.

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