Isolation Of PCR Products

实验概要

Rapid and efficient purification of PCR products from salts, primers, dNTPs, and other non-nucleic acid reagents.

实验原理

The ChargeSwitch^® Technology

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

ChargeSwitch^® Purification Buffer (N5)

ChargeSwitch^® Magnetic Beads

ChargeSwitch^® Wash Buffer (W12)

ChargeSwitch^® Elution Buffer (E5)

实验材料

PCR samples

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 

96-Well Magnetic Separator 

Shaker

实验步骤

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.

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