High Throughput Isolation Of PCR Products Using ChargeSwitch® PCR Clean-Up
实验概要
The ChargeSwitch® PCR Clean-Up Kit allows rapid and efficient purification of PCR products from salts, primers, dNTPs, and other non-nucleic acid reagents. The kit is designed for automated processing of large numbers of samples in 96-well plates using a liquid handling robot. The kit is designed for the purification of DNA fragments ranging from 90 bp-40 kb and the purified PCR product is suitable for any downstream applications of choice.
The PCR product is purified in less than 10 minutes with the ChargeSwitch® Technology using an automated liquid handling robot. For more information on the Charge Switch® Technology, see below.
Use of the ChargeSwitch® PCR Clean-Up Kit has been demonstrated on the Tecan Genesis® robotic workstation to purify PCR products in a fully automated system from large numbers of samples in a 96-well format. Other liquid handling robots are suitable provided that each is equipped with a gripper arm, a 96-well magnetic separator, and other additional hardware as described below. This manual provides general guidelines and a protocol that can be used to develop a script for your robot.
实验原理
The ChargeSwitch® Technology is a novel magnetic bead-based technology providing a switchable surface that is charge dependent on the surrounding buffer pH to facilitate nucleic acid purification. The ChargeSwitch® chemistry is ideal for purification of DNA using liquid handling robots, avoiding the need for centrifugation steps or the use of ethanol or chaotropic salts. In low pH conditions, the ChargeSwitch® Magnetic Beads have a positive charge and binds the negatively charged nucleic acid backbone (see figure below). Proteins and other contaminants are not bound and are washed away using the wash buffer. To elute nucleic acids, the charge on the surface is neutralized by raising the pH to 8.5 using a low salt elution buffer (see figure below). Purified DNA elutes instantly into this elution buffer.
主要试剂
The components supplied in the ChargeSwitch® PCR Clean-Up Kit are listed below. The reagents supplied are sufficient to perform 960 purifications.
Note: Some reagents maybe supplied in excess in the amount needed.
Product Contents | |
ChargeSwitch® Magnetic Beads (25 mg/ml in 10 mM MES, pH 5.0, 10 mM NaCl, 0.1% Tween 20) | 10 ml |
ChargeSwitch® Purification Buffer (N5) | 58 ml |
ChargeSwitch® Wash Buffer (W12) | 300 ml |
ChargeSwitch® Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5) | 48 ml |
主要设备
96 x 200 µl U-bottomed microtiter plate
Any liquid handling robotic workstation with a gripper arm to process samples in 96-well plates.
Appropriate tips for liquid dispensing and aspiration [You may use any tips of choice to dispense and aspirate liquid during the purification procedure. Consider the following when choosing an appropriate tip to use. 1. Fixed vs. disposalbe tips. 2. Tip size vs. head size. 3. Conductive or non-conductive. 4. Sterile or non-sterile. 5. Filtered or non-filtered]
96-Well Magnetic Separator [The 96-Well Magnetic Separator available from Invitrogen (cat. no. CS15096) is a magnetic separation rack suitable for use in protocols with magnetic beads. The rack can hold up to 96 samples in a deep well plate. The deep well plate fits onto the magnetic separator, associating the array of 24 neodymium magnets with 96 samples for magnetic sample processing (see figures below).]
Shaker
实验材料
PCR samples
实验步骤
Automated Procedure for Purifying PCR Products
Automated Protocol
This section provides a general protocol for automated purification of PCR products in a 96-well format. Use the parameters and guidelines provided above, as well as the protocol below to develop the script for your liquid handling robot. For more information, see http://www.invitrogen.com or call Technical Service.
Follow the protocol below to purify PCR products. The volumes given are on a per sample basis.
1. To ~50 µl PCR samples in 96-well plates, add 10 µl ChargeSwitch® Magnetic Beads.
2. Add 60 µl Purification Buffer (N5).
3. Shake at medium speed for 30 seconds to evenly distribute the magnetic beads in the solution.
4. Move samples to the 96-Well Magnetic Separator.
5. Wait for 30 seconds.
6. Aspirate all of the supernatant and discard, leaving behind the pellet of beads.
7. Move samples to the shaker.
8. Add 150 µl Wash Buffer (W12).
9. Shake at medium speed for 30 seconds to evenly distribute the magnetic beads in the solution.
10. Move samples to the 96-Well Magnetic Separator.
11. Wait for 1 minute.
12. Aspirate all of the supernatant and discard, leaving behind the pellet of beads.
13. Move samples to the shaker.
14. Add 150 µl Wash Buffer (W12).
15. Shake at medium speed for 30 seconds to evenly distribute the magnetic beads in the solution.
16. Move samples to the 96-Well Magnetic Separator.
17. Wait for 1 minute.
18. Aspirate all of the supernatant and discard, leaving behind the pellet of beads.
19. Move samples to the shaker.
20. Add 50 µl Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5).
21. Shake at fast speed for 1 minute to evenly distribute the magnetic beads within the solution.
22. Wait for 30 seconds.
23. Move samples to the 96-Well Magnetic Separator.
24. Wait for 1 minute.
25. Slowly aspirate supernatant containing the purified PCR product to a 96 x 200 µl U-bottomed microtiter plate.
Storage
Store the purified PCR product at –20°C or use the PCR product in the downstream applications of choice.
注意事项
Safety Information
Follow the safety guidelines below when using the ChargeSwitch® Kit.
Treat all reagents supplied in the kit as potential irritants.
Always wear a suitable lab coat, disposable gloves, and protective goggles.
If a spill of the buffers occurs, clean with a suitable laboratory detergent and water. If the liquid spill contains potentially infectious agents, clean the affected area first with laboratory detergent and water, then with 1% (v/v) sodium hypochlorite or a suitable laboratory disinfectant.
Recommendations: 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 bead pellet.
Resuspend the ChargeSwitch® Magnetic Beads thoroughly before use.
When removing supernatant, aspirate slowly to ensure that the pellet of beads is not disturbed.
To maximize DNA yield, make sure that all Wash Buffer is removed before elution and the beads are fully resuspended during the elution step.
