Fast Direct PCR from Mouse Tail Samples

Authors

highQu

Summary

Mouse genotyping or similar applications are frequently time-consuming due to tedious template purification procedures before the PCR. SampleIN™ Direct PCR Kit is a premium tool for a fast direct PCR eliminating the need of template purification. The kit is an excellent choice for direct PCR from mouse tail or ear, mammalian tissues, hair follicle, buccal swabs and blood.

• Image 1 Results with SampleIN™ Direct PCR Kit

Introduction

The protocol describes fast and simple approach for mouse genotyping without the template purification.

Rapid 15 min DNA extraction using DPK Lysis and Protease Buffers in a single tube generates PCR template extract which is further amplified under fast cycling conditions with a hot-start Taq master mix that includes red dye for direct gel loading.

The ALLin™ HS Red Taq Mastermix includes a hot start Taq DNA Polymerase what ensures high yield, highly specific low background amplification. Mix components allow for a fast PCR cycling and increase success when working with complex templates or multiplexing.

Materials

1. SampleIN™ Direct PCR Kit (highQu), includes following components:

a) DPK Lysis Buffer, 5X  - it contains all components required for an efficient lysis of mammalian tissue samples.

b) DPK Protease Buffer, 10X  - contains proteases to eliminate sample proteins.

c) 2X ALLin™ HS Red Taq Mastermix  - it contains hot-start enzyme, dNTPs, MgCl2, enhancers, stabilizers, red electrophoresis tracking dye and density reagents for gel loading.

2. Mouse tail samples (2 mm or 3-5 mg each)

3. PCR Water

Procedure

1. Sample DNA extraction procedure:

• Take typical measures to prevent contamination, keep your bench clean, wear gloves, and use sterile tubes.
• Thaw DPK Buffers at room temperature. Mix well before use.
• Prepare a 100 µl extraction reaction in a sterile vial:

1. Add 2 mm or 3-5 mg of mouse tail sample into the tube.
2. Add 20 μl of DPK Lysis Buffer, 5X into the vial with the sample.
3. Add 10 μl of DPK Protease Buffer, 10X into the vial.
4. Add 70 μl of PCR Water.
5. Mix gently and place into the thermal block/water bath set like: 75°C - 5 min for lysis. Vortex twice during lysis.
6. Inactivate proteases at 95°C - 10 min.
7. Add 900 µl of PCR Water into the sample.
8. Centrifuge 1 min to pellet cell debris.
9. Remove supernatant into the new sterile tube.

• Store the crude sample at -20°C for several months or use immediately for PCR.

2. Perform PCR reaction:

• Include a no-template control and positive control in parallel.
• Thaw and keep PCR reagents on ice. Mix well before use.
• Add following into the PCR tube for each 25 µl reaction.

1. Rev. & For. Primers: 0.1-0.4 µM final each (≤ 2 µl of 10 µM)
2. Template: 1-5 µl of extraction supernatant from step 1.
3. PCR Water: to 25 μl
4. ALLin™ HS Red Taq Mastermix, 2X: 25 µl

• Mix gently. Place into the PCR instrument set like:

1. Initial denaturation: 1 cycle: 95°C - 2 min
2. Denaturation 40 cycles: 95°C - 15 sec
3. Annealing 40 cycles: 55-65°C – 15 sec
4. Extension 40 cycles: 72°C – 15 sec/kb (90 sec. for multiplex)

• Analize the PCR products on the gel, they are ready for loading directly after the PCR. In a 2% agarose TAE gel the red dye migrates with~350 bp DNA, in 1% agarose TAE gel with ~ 600 bp DNA fragments.
• Store probes for short time on ice, for longer period at -20°C.

Anticipated results

• Ready-to load PCR in 50 minutes without template purification
• Single-tube 15 min DNA extraction combined with fast hot-start PCR
• Red dye in the PCR master mix for direct gel loading
• High yields under standard or fast cycling conditions
• Success with GC/AT rich templates

Further details

Download the SampleIN™ Direct PCR Kit User Manual

References

http://www.highqu.com/products/pcr-qpcr/standard-and-direct-pcr/samplein-direct-pcr-kit.html

Documents for download

• DPK01_SampleIN_Direct_PCR_Kit_PI.pdf

Stats

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.