AutomatedGenomicDNAExtraction

實驗概要

This section  provides a general protocol for automated isolation of genomic DNA from  10-20 μl blood samples in a 96-well format using the ChargeSwitch^? 20μl blood kit (CS11010-10). Use this general protocol to develop the script for your liquid handling robot.

主要試劑

ChargeSwitch? Lysis Buffer (L12)

Proteinase K

ChargeSwitch? Magnetic Beads

ChargeSwitch? Purification Buffer (N5)

ChargeSwitch? Wash Buffer (W12)

ChargeSwitch? Elution Buffer (E5) or TE Buffer (not supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)

主要設備

Liquid handling robot configured to process samples in 96-well plates

96 x 2 ml deep well plate(s)

96 x 300 μl U-bottomed microtiter plate

實驗材料

10-20 μl blood samples

實驗步驟

Before Starting
Perform the following before beginning:

• Prepare Lysis Mix: For each sample, mix 0.5 ml of ChargeSwitch^?  Lysis Buffer (L12) and 5 μl of Proteinase K to prepare the Lysis Mix.  Scale up the volume of reagents used (based on number of samples) to  prepare a master mix.

• Prepare Purification Mix: For each sample, mix 50 μl of ChargeSwitch^? Purification Buffer (N5) and 20 μl of ChargeSwitch^?  Magnetic Beads (make sure that the beads are thoroughly resuspended) to  prepare the Purification Mix. Scale up the volume of reagents used  (based on number of samples) to prepare a master mix.

Automated Protocol
Follow the protocol below to isolate genomic DNA from 10-20 μl blood samples. The volumes given are on a per sample basis.

1. Start with 96 x 10-20 μl blood samples in a 96 x 2 ml deep well plate.

2. Add  500 μl of Lysis Mix and incubate at room temperature for 10 minutes.  Once during the incubation, pipet up and down gently 15 times to mix.  Set the pipette tip to 350 μl and avoid forming bubbles.

3. Add 70 μl of Purification Mix (make sure that the beads are thoroughly resuspended)

4. Shake at medium fast speed (e.g. pulse, 10 seconds) to evenly distribute the magnetic beads within the solution.

5. Shake samples rapidly for 20 seconds to mix.

6. Wait for 30 seconds.

7. Move samples to the 96-Well Magnetic Separator.

8. Wait for 90 seconds.

9. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.

10. Remove samples from the 96-Well Magnetic Separator.

11. Add 500 μl of ChargeSwitch^?  Lysis Buffer (L12; no Proteinase K) and shake samples rapidly for 20  seconds to evenly distribute the magnetic beads within the solution.

12. Add 50 μl of ChargeSwitch^? Purification Buffer (N5) and shake at medium speed for 20 seconds to mix. Samples should appear clear, with no brown flecks.

13. Wait for 30 seconds.

14. Move samples to the 96-Well Magnetic Separator.

15. Wait for 60 seconds.

16. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.

17. Remove samples from the 96-Well Magnetic Separator.

18. Add 500 μl of ChargeSwitch^? Wash Buffer (W12).

19. Shake at medium speed (e.g. pulse, 10 seconds) to evenly distribute the magnetic beads within the solution.

20. Move samples to the 96-Well Magnetic Separator.

21. Wait for 60 seconds.

22. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.

23. Leave samples on the 96-Well Magnetic Separator for the second wash.

24. Add 500 μl of ChargeSwitch^? Wash Buffer (W12).

25. Wait for 30-60 seconds.

26. Slowly aspirate all of the supernatant and discard, leaving behind the pellet of beads.

27. Move samples to the shaker.

28. Add 100 μl of Elution Buffer. Pipet up and down gently 50 times to mix (set the pipette tip to 75 μl).

29. Shake rapidly for 1-2 minutes to completely disperse the beads within the solution.

30. Move samples to the 96-Well Magnetic Separator.

31. Wait for 1 minute.

32. Slowly aspirate supernatant containing the DNA to a 96 x 300 μl U-bottomed microtiter plate.

Storing DNA
Store the purified DNA at -20°C or use immediately for downstream analysis. Avoid repeatedly freezing and thawing DNA.
Quantitating DNA Yield
To quantitate yield of your DNA, use the Quant-iT? PicoGreen^? dsDNA Quantitation Kit (Catalog no. P7589).

注意事項

To maximize DNA yield, follow these recommendations when processing your samples:

• Ensure that the robotic tips enter the wells of the plates without interfering with the pellet of beads.

• When  removing supernatant, leave samples on the 96-Well Magnetic Separator  and aspirate slowly to ensure that the pellet of beads is not disturbed.

• When resuspending pelleted ChargeSwitch^? Magnetic Beads, make sure that all beads are fully resuspended to maximize DNA recovery.

• To maximize DNA yield, make sure that all Wash Buffer is removed before elution.

• To maximize DNA yield, make sure that the beads are fully resuspended during the elution step.