Zebrafish genotyping

ZEBRAFISH GENOTYPING

Genomic DNA (gDNA) extraction:

1. Transfer 45 µl of lysis buffer (see recipe below) into PCR tubes, on ice.

2. Transfer processed or fresh zebrafish tissue into prepared PCR tube, 1 sample per tube.

3. Incubate tubes at 98 °C for 10 minutes to lyse tissue, cool to 20 °C.

4. Add 5 µl of 10 mg/ml Proteinase K to each PCR tube.

5. Incubate at 55 °C for 12 hours, then 98 °C for 10 minutes to deactivate the proteinase K.

6. Store gDNA at 4 °C or -20 °C for short or long-term use, respectively. Use 2.5 µl of gDNA per PCR reaction (pull from top of tube to avoid tissue debris).

Genotyping PCR and digest:

7. Prepare genotyping master mix using your favorite standard PCR reagents (see recipe below) with enough for 22.5 µl per reaction, distribute to PCR tubes. 

8. Add 2.5 µl of gDNA per sample to each tube.

9. Standard genotyping PCR: 

10. Use 5 µl of PCR product and digest using the indicated NEB® restriction enzyme and digest conditions (25 µl reaction).

Genotyping gel:

11. Prepare a 3% agarose DNA gel in 1x TAE (5 µl Ethidium Bromide per 100 ml of 1x TAE).

12. Load half of the restriction digest + dye per sample into corresponding well in the gel.

13. Run gel at 100V for half an hour, or until separation of WT and mutant bands is sufficient to call the genotype (see below for mpp6b).

Solutions:

2x PCR Buffer (Store at Room Temp)

2x gDNA Lysis Buffer (Store at Room Temp)

*NP40 is also known as IGEPAL CA-630

PCR Master Mix (Promega GoTaq® DNA polymerase, prepare on ice)

Mpp6b genotyping gel:

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