AutomatedExtractionNormalizedDNABuccalKit

实验概要

This section  provides a general protocol for automated isolation of genomic DNA from  human buccal cell swabs in a 96-well format using the ChargeSwitch^®gDNA  Normalized Buccal Cell Kit (Catalog no. 11020-10). Use this general  protocol to develop the script for your liquid handling robot.

主要试剂

1. ChargeSwitch^® Lysis Buffer (L11)

2. Proteinase K
3. ChargeSwitch^® Magnetic Beads

4. ChargeSwitch^® Purification Buffer (N6)
5. ChargeSwitch^® Wash Buffer (W12)

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

主要设备

1. Liquid handling robot configured to process samples in 96-well plates
2. 96 x 2 ml deep well plates
3. 96 x 300 µl U-bottomed microtiter plate
4. Optional: 96 x 2 ml glass-filled, polypropylene, Unifilter^® Microplate (Whatman, Catalog no. 7720-7235)   

实验材料

Buccal swabs

实验步骤

Before Starting

Perform the following before beginning:

• Prepare Lysis Mix: For each sample, mix 1 ml of ChargeSwitch^®  Lysis Buffer (L11) and 10 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 100 µl of ChargeSwitch^® Purification Buffer (N5) and 40 µl of ChargeSwitch^®  Magnetic Beads to prepare the Purification Mix (make sure that the  beads are thoroughly resuspended). Scale up the volume of reagents used  (based on number of samples) to prepare a master mix.

Automated Protocol

Follow this protocol to isolate genomic DNA from buccal swabs. The volumes given are on a per sample basis.

1. Start with 96 buccal cell samples in a 96 x 2 ml deep well plate or 96 x 2 ml filter plate.

2. Add 1 ml of Lysis Mix (see above) and incubate at 37°C for 20 minutes (use a heating block).

Note: Optimal incubation parameters (i.e. time) vary depending on the  sample and automation plasticware, and should be determined  empirically.

3. After incubation, transfer or filter as appropriate, as much of  the lysate as possible to a 96 x 2 ml deep well plate, without  interfering with the samples.

4. Add 140 µl of Purification Mix (see above; make sure that the beads are thoroughly resuspended).

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

6. Wait for 10 seconds.

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

8. Wait for 60-90 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^® Wash Buffer (W12).

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

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

14. Wait for 60-90 seconds.

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

16. While samples are still on the 96-Well Magnetic Separator, add 500 µl of ChargeSwitch^® Wash Buffer (W12).

17. Wait for 30-60 seconds.

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

19. Move samples to the shaker.

20. Add 150 µl of ChargeSwitch^® Elution Buffer (E5).

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

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

23. Wait for 1 minute.

24. 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  applications such as PCR. Avoid repeatedly freezing and thawing DNA.

Quantitating DNA Yield

To quantitate the yield of your DNA, use the Quant-iT™ DNA Assay Kit, High Sensitivity (Catalog no. Q33120).

注意事项

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.

Using a Filter Plate

Some of the Lysis Mix may be absorbed by the sample, resulting in a  lower volume of lysate being available for purification. To maximize  recovery volume and minimize contaminant transfer during lysate  preparation, you may prepare lysates in a 96 x 2 ml filter plate, then  directly filter the supernatant into a 96 x 2 ml deep well plate to  perform the remainder of the purification procedure. We recommend using  the 96 x 2 ml glass-filled polypropylene Unifilter^® Microplate from Whatman (Catalog no. 7720-7235). Other 96 x 2 ml filter plates are suitable.