TITLE: Ventral Midbrain Genomic PCR
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
This protocol details ventral midbrain genomic PCR.

[STEPS]
SECTION: Brain tissue collection
1. Euthanize mouse via cervical dislocation.
SECTION: Brain tissue collection
2. Isolate   coronal midbrain section using stainless steel brain matrix (Stoelting 51386).

SECTION: Brain tissue collection
2.1. Remove cortex and dorsal midbrain tissue.
SECTION: Brain tissue collection
2.2. Separate ipsilateral and contralateral ventral midbrain regions.
SECTION: Brain tissue collection
2.3. Immediately freeze on dry ice and store tissue at -80 °C until DNA extraction.

SECTION: DNA extraction from frozen brain tissue
3. Use Qiagen DNeasy Blood and Tissue Kit (Cat 69504).
SECTION: DNA extraction from frozen brain tissue
4. Equilibrate ventral midbrain tissue to Room temperature.

 

SECTION: DNA extraction from frozen brain tissue
5. Cut tissue into small pieces.
SECTION: DNA extraction from frozen brain tissue
5.1. Use a pipette tip to transfer tissue to a sterile 6cm dish.
SECTION: DNA extraction from frozen brain tissue
5.2. Add 180 µL of Buffer ATL to the tissue.

SECTION: DNA extraction from frozen brain tissue
5.3. Dice up tissue with sterile scalpel.
SECTION: DNA extraction from frozen brain tissue
5.4. Transfer tissue and buffer to 1.5mL etube.
SECTION: DNA extraction from frozen brain tissue
6. Add 20 µL Proteinase K to each sample.

SECTION: DNA extraction from frozen brain tissue
6.1. Mix thoroughly by vortexing.
SECTION: DNA extraction from frozen brain tissue
6.2. Incubate at 56 °C until samples are completely lysed.

 

SECTION: DNA extraction from frozen brain tissue
7. Add 4 µL RNase A (100 1629) to each sample.

SECTION: DNA extraction from frozen brain tissue
7.1. Mix by vortexing.
SECTION: DNA extraction from frozen brain tissue
7.2. Incubate at Room temperature for 2 min.

SECTION: DNA extraction from frozen brain tissue
8. Mix Buffer AL with ethanol 1:1.
SECTION: DNA extraction from frozen brain tissue
8.1. Add 400 µL Buffer AL / ethanol mix to each sample.

SECTION: DNA extraction from frozen brain tissue
8.2. Vortex for 15 s.

SECTION: DNA extraction from frozen brain tissue
9. Transfer samples to DNeasy Mini spin columns (placed in 2mL collection tubes).
SECTION: DNA extraction from frozen brain tissue
9.1. Centrifuge at ≥6000 x g for 1 min.

SECTION: DNA extraction from frozen brain tissue
9.2. Discard flow through.
SECTION: DNA extraction from frozen brain tissue
10. Transfer column to new collection tube.
SECTION: DNA extraction from frozen brain tissue
10.1. Add 500 µL Buffer AW1.

SECTION: DNA extraction from frozen brain tissue
10.2. Centrifuge at ≥6000 x g for 1 min.

SECTION: DNA extraction from frozen brain tissue
10.3. Discard flow through and collection tube.
SECTION: DNA extraction from frozen brain tissue
11. Place the column in a new collection tube – add 500 µL Buffer AW2.

SECTION: DNA extraction from frozen brain tissue
11.1. Centrifuge 20000 x g for 3 min to dry the column.

SECTION: DNA extraction from frozen brain tissue
11.2. Carefully remove the column to avoid contamination with residual ethanol.
SECTION: DNA extraction from frozen brain tissue
11.3. If the column touches ethanol flow through, spin again in new collection tube for 1 min.

SECTION: DNA extraction from frozen brain tissue
12. Place the column in clean 1.5mL etube – add 150 µL of elution buffer (Buffer AE).

SECTION: DNA extraction from frozen brain tissue
12.1. Incubate at Room temperature for 1 min.

SECTION: DNA extraction from frozen brain tissue
12.2. Centrifuge at ≥6000 x g for 1 min to elute DNA.

SECTION: Genomic PCR
13. DNA concentration measured using a NanoDrop One Spectrophotometer (Thermofisher Scientfiic).
SECTION: Genomic PCR
13.1. All samples diluted to 25 1909 with Buffer AE.

SECTION: Genomic PCR
14. Use the Kapa2g Fast HotStart PCR Kit (Roche 07960930001) according to the manufacturer’s instructions.

 
 
 

PCR Cycle:
 


SECTION: Genomic PCR
15. Run samples on 2% Agarose gel at 100V for 35-45 minutes.