Kit, one-step reaction using the single buffer system

llele-in-One mouse tail direct PCR buffer releases DNA from mouse tails for genotyping PCR. A onestep reaction using the single buffer system is sufficient for preparing DNA as PCR template; phenol extraction, precipitation, or any further purification is not necessary. The buffer contains a combination of enzyme(s), detergents, and other chemical reagents that will lyse the mouse tail tissues or other tissues without destroying DNA. An aliquot is then directly used as template in a genotyping PCR reaction. It is best used in combination with Allele-in-One PCR MasterMix (Allele-in-One Mouse Tail Direct PCR Kit, ABP-PP-MT02###), specifically formulated to accommodate DNA template from lysis buffer that contains proteases. This buffer can also be used to prepare genomic DNA for direct PCR from other tissues, such as mouse ear, rat tissues, etc. Slight adjustment and optimization may be needed for each type of tissue. All -in-One Mouse Tail Direct PCR system offers a variety of advatages including:
ele

Allele-In-One Mouse Tail Direct PCR System w/ Purification

plus purification for preparing DNA as PCR template.
Box 1 | Genotyping Results

Type I Type II

Mouse tail biopsy at ~0.3cm was incubated in 100L Lysis Buffer for 2 hours at 55oC. Genomic DNA in 1ml lysate was directly amplified in a 10L PCR reaction (5mL of 2X PCR Mastermix added) for 29 cycles.
Each batch of reagents is vigorously tested for consistency and stability.

Storage: -20C and -80C

Allele-in-One Mouse Tail buffer is suitable for many applications such as: Lysis of mouse tail samples for genotyping PCR Lysis of other sample types (e.g. mouse ear, yolk sac or tissue culture cells). The Mouse Tail Direct PCR Kit includes Allele-in One PCR MasterMix, which is optimized for DNA template prepared with the lysis buffer but can also be used for most routine PCR reactions.

Protocols
1. Prepare 0.3 cm mouse tail biopsy sample. 2. Combine 100 l Lysis Buffer with biopsy sample in 1.5mL reaction tube. 3. Incubate at 50-55C, with or without rotation, for 30 min or overnight. 4. Purify lysate using PG columns, binding buffer (AB), and wash buffer(AW). Elute the DNA using the elution buffer (TE) into 1.5ml recovery tubes. 5. For each new PCR primer pair, use 0.05 to 0.5L of the lysate as PCR template while optimizing the PCR conditions. Once determined, the volume from the tissue lysate will be typically between 0.2 and 0.5L. This amount should be used in all future reactions with the same primer pair. 6. 2X PCR MasterMix should be added as half of the final reaction volume. Please see reverse side for Recommended PCR Protocol & Troubleshooting

Simple buffer system: Unique from Allele Biotech Enzymes, this single buffer system is the only product that contains everything for lysis, no need to add protease K or any other components before use. Single step reaction: The incubation is to be performed at a convenient 50-55C temperature for 0.5 hours or longer, and the lysate is ready for PCR. Consistent: Works with extremely high success rates and is fully guaranteed if used in combination with Allele-in-One PCR MasterMix system. Lowering costs: Adds only 40 cents or less to each genotyping sample while saving enormous amounts of material and time from traditional methods.

Box 2 | Product List

MouseTail Direct PCR Buffer w/ Purification 100l per reaction
ABP-PP-MT03100 ABP-PP-MT03250 100 rxns 250 rxns

Mouse Tail Direct PCR w/ Purification Kit 10l per reaction*

ABP-PP-MT04100 ABP-PP-MT04250 100 rxns 250 rxns

Mastermix, also sold separately: Cat #: ABP-PP-MM02961

*All Mouse Tail PCR Kits contain 2X PCR

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Protocolsand Troubleshooting
Lysate Purification Protocol:
Important Reminders Add ethanol to AW buffer, wash buffer. All centrifugation steps should be performed at ~14k rpm.

Box 4 | Purification Kit Contents

Component Allele PG Column 2 mL collection tubes 1.5 mL elution recovery tubes AB: Binding buffer AW: Washing buffer* TE: TE buffer 100 rxns 100 100 100 35 mL 25 mL 10 mL 250 rxns 250 250 250 87.5 mL 62.5 mL 35 mL

1. 2. 3. 4. 5. 6. 7. 8. tube. 9. 10. 11.

Add an equal volume of AB buffer to the Lysate. Check that the Allele PG column is placed inside 2 Transfer Lysate/AB mix to the Allele PG column. Centrifuge for 1 minute and discard flow-through Wash DNA by adding 700 L AW buffer. Centrifuge for 1 minute and discard flow-through Add an additional 500 L AW buffer and centrifuge

mL collection tube.

inside collection tube. *Note: All volumes for AW (washing buffer) indicate volumes before the addition of 95-100% ethanol. For any (100) reaction/prep kit add mL, and for any (250) reaction/ prep kit add 250 mL 95-100% ethanol. Trouble Shooting: 1) No PCR products or inconsistent results from time to time: Most of the time this is caused by too much genomic DNA. This inhibits the PCR reaction, especially from samples that are prepared by overnight incubation. Try using less lysate as PCR template. Another explanation may be because of Insufficient lysis. Occasionally the age of the animal or how the tails have been stored determine if it requires a longer incubation at a higher temperature or in a larger volume of lysis buffer (e.g. 200 l for mice older than 3 months). 2) Smeared DNA bands: This may be caused by too much debris from lysed tissue, which interferes with the Taq polymerase. Add a 3-10 min centrifuge step using a microcentrifuge at top speed and transfer the upper portion of the lysate to PCR reactions. Also too much time between setting up and starting PCR reactions may cause Taq to degrade. Possible improvements include: Setting PCR contents on ice and moving quickly to a PCR machine or heating the lysate for 15 min at >80C after the 50-55C incubation before using as PCR template.

collected inside the collection tube. for 1 minute. Transfer Allele PG column to clean microcentrifuge Elute DNA by adding 30-50 L TE buffer directly to Incubate at room temperature for 1 minute and Store DNA at -20C.

membrane of Allele PG column. centrifuge for 1 minute.

Box 3 | Recommended PCR Setup

Allele-in One PCR 2X MasterMix Lysate Forward Primer Reverse Primer 5L 0.05 - 0.5L** 0.2 - 0.5L** 0.2 - 0.5L**

Fill to 10L with nuclease-free water **Lysate and primer concentrations vary based on primer pair.

Related Products
Custom Oligo Synthesis We specialize in very long oligos (e.g., 215 nt) and will ship or deliver our oligos within 24hours. Our oligo synthesis service offers a large variety of synthesis scales, modifications and purifications for our customers various needs. Allele-In-One PCR MasterMix Pre-mixed solution containing: Taq DNA polymerase, MgCl2, dNTPs, an inhibitor of Taq at low temperatures, and optimal PCR reaction buffers.

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