Editing Blood RT-PCR (section)

== Extract DNA from&nbsp; Whatman S&S 903&nbsp; ==

Spot 50 ul blood on each on a Whatman S&S 903 card allow to dry for 4 h at room temperature. Place filter paper punch in a microcentrifuge tube Soak in 65 μL of TE buffer Incubate at 50°C for 15 minutes. Press the punches to at the bottom of the tube several times, using a new pipette tip for each punch Heat at 97°C for 15 minutes to elute DNA

'''FTA Purification Reagent (Fitzco, Inc) 500 ml:'''

0.29 g NaCl; 

5 ml 1 M Tris pH 7.5

1 ml 0.5 M EDTA

2.5 ml Triton X-100

'''RBC lysis buffer:&nbsp;1X &nbsp;ACK (Ammonium Chloride Potassium)'''

&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 155mM NH4Cl &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;8.29g

&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 10mM KHCO3 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;1.0g

&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 0.1mM EDTA &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;0.2 ml (500mM)&nbsp;







10X&nbsp;'''RBC lysis buffer'''<br/>NH4Cl 80.2 g<br/>NaHCo3 sodium bicarbonate 8.4 g<br/>EDTA disodium 3.7 g<br/>Add water to 1 L, store at 4 oC for 6 months



5 ul of whole blood one can expect between 35,000 and 50,000 white blood cells; each cell containing approximately 7 pg of genomic DNA Taking the upper limit, 350 ng of total genomic DNA is expected from 5 ul of whole blood.

Preparing an FTA Disc for DNA Analysis

1. Take two sample discs from the dried spot For plant samples a 2.0mm disc is recommended.

2. Place discs in the bottom of a spin basket insert.

3. Place the spin basket into a 2.0mL micro centrifuge tube.

4. Add 500µL FTA purification reagent to the basket.

5. Incubate for 1 minute at room temperature.

6. Centrifuge at 6000 x g for 30 seconds.

7. Remove the basket and decant used reagent.

8. Return the basket to the micro centrifuge tube and repeat Steps 4-7 twice for a total of 3 washes with the FTA purification reagent.

9. Add 495µL FTA purification reagent and 5µL Proteinase K (10mg/mL) to the FTA discs in the basket.

10. Incubate for 1 hour at 60°C to remove residual Histones.

11. Centrifuge at 6000 x g for 30 seconds.

12. Remove the basket and decant used reagent/buffer.

13. Return the basket to the micro centrifuge tube.

14. Add 500µl of TE-1 buffer to the tube.

15. Centrifuge at 6000 x g for 30 seconds.

16. Remove the basket and decant used buffer.

17. Return the basket to the micro centrifuge tube.

18. Repeat steps 14-17 twice for a total of 3 washes with TE-1 buffer.

19. Transfer discs to a new tube.

20. Ensure that all the liquid has been removed before performing analysis.

TE Buffer (10 mM Tris-HCL, 0.1 mM EDTA, pH 8.0) Solution 1: 0.1N NaOH, 0.3mM EDTA, pH 13.0 Solution 2: 0.1M Tris-HCL, pH 7.0