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Mouse Direct PCR Kit (For Genotyping)

For research use only.

The Mouse Direct PCR Kit provides a fast preparation and PCRamplification that is specifically designed for mouse genotyping.

Selleck's has been cited by 68 publications

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Description

The Mouse Direct PCR Kit provides a fast preparation and PCRamplification that is specifically designed for mouse genotyping. TheBuffer L and Protease Plus rapidly digest mouse tail, ear and toe to release intact genomic DNA that can be used directly as the templatefor PCR amplification.By using this kit, the digestion process only takes 15 minutes. In addition, the 2x M-PCR OPTI Mix(which includes an optimized Taq Polymerase) ensures highamplification efficiency of target DNA.

1. Comparison Between Different Methods

Reagent	Price ($)	Genotyping Method
Reagent	Price ($)	NaOH-HCL	Proteinase K	Selleck Direct PCR Kit
Lysis Buffer	Sigma, 0.010	+	+	Buffer L
Proteinase K	Sigma P6556, 1U/reaction, 0.034		+	Protease Plus
Phenol Chloroform	Sigma P3803, 200μL/reaction, 0.116	+	+
Alcohols	Sigma I9030, 459836, 0.084	+	+
Taq	NEB M0273X, 1U/reaction, 0.122	+	+	2 x M-PCR OPTI^TM Mix
dNTP	Sigma GE28-4065-64, 0.25mM, 0.066	+	+
MgCL₂, DNA Loading	Sigma, 0.010	+	+
Price ($) /Reaction		0.41	0.44	0.39

2. Comparison of PCR results (wild-type mouse)

Lane 1-10 represents different PCR primers. Two of them serve as the negative controls (Lane 9 and 10) which only display bands when using the transgenic mouse sample. Therefore, Mouse Direct PCR kit ensures a higher PCR specificity, making it a reliable product for scientific research.

3. Advantages

Analysis of Result:
The Mouse Direct PCR Kit applies to the preparation for samples from mouse tail, ear and toe. No significant difference was observed between the groups, which, digests in 55℃ for 15 min or 30 min. The kit is also effective for the amplification of large fragments (e.g. 3Kb).
Note: The primers for amplification of 500bp and 3Kb-fragments is designed based on the mouse GAPDH gene, while the primers for 1Kb and 2Kb-fragments is designed based on the mouse Ppip5k2 gene.

4. Components

Complete Kit	B40013 (200 rxns)	B40015 (500 rxns)
Buffer L	20 mL	25 mL x 2
Protease Plus	0.4 mL	1 mL
2 x M-PCR OPTI^TM Mix (Dye Plus)	1 mL x 2	1 mL x 5
User Guide	1	1

Buffer L: Lysis buffer.

Protease Plus: For rapid and efficient digestion of mouse tissue in only 15 minutes!

2x M-PCR OPTI^TM Mix: Includes Selleck's trademarked and optimized Taq DNA polymerase, dNTPs, MgCl₂, and reaction buffer.

Storage (From the date of receipt)

2 x M-PCR OPTI^TM Mix and Protease Plus should be stored at -20°C for 2 years. If the PCR Mix is to be used frequently, it can be stored at 4°C for up to 10 days.

Buffer L should be stored at 4°C.

Protocol

1. Experimental Protocol

1) Genomic DNA Preparation
Place the mouse tail, ear, or toe in a 1.5 mL centrifuge tube. Thoroughly mix 100 μL of fresh Buffer L with 2 μL of Protease Plus for a single sample in a separate tube. Add the protease mixture to the mouse tissue tubes with the tissue cut submerged in it, then incubate at 55°C for 15 minutes. After the digestion process, incubate at 95°C for 5 minutes to inactivate protease. The tissue lysate can now be used as a PCR template.

2) PCR Genotyping
Add dd H₂O, primers, template, and 2 x M-PCR OPTI^TM Mix into a PCR tube according to the recommended concentrations. Give the mixture a quick spin in the centrifuge and load into PCR amplifier to begin amplification. It is recommended to prepare PCR reaction in ice-bath.

PCR Reaction Components	20 μL Reaction Volume (μL)	50 μL Reaction Volume (μL)
ddH₂O	8	21
Forward Primer (10 μM)	0.5	1
Reverse Primer (10 μM)	0.5	1
Template	1	2
2 x M-PCR OPTI™ Mix	10	25

PCR Steps	Temperature (°C)	Time	Cycles
1	94	5 min	1
2	94	20 sec	35
3	50-65	30 sec
4	72	X min (2 kb /min)
5	72	5 min	1
6	12	--	1

Note: Before First Use
1. All reagents in this kit have been optimized for use together and any modifications or alternative uses are prohibited.
2. For each step, make sure every reagent in the kit is blended well prior to use.
3. During the tissue digestion step, shaking the tubes 1-2 times will be helpful to release the genomic DNA.
4. For most mouse tissue samples, an incubation of 15 minutes at 55°C will suffice for genomic DNA extraction. The tissue may still appear intact, but extraction has occurred.
5. The extracted mouse genomic DNA should be applied immediately prior to the PCR amplification step. Inappropriate long-term storage may cause unreliable PCR amplifications.

Trouble Shooting

Please review the following for trouble-shooting options when you encounter technical difficulties. Alternatively, feel free to contact Selleck technical support directly.

Problem	Potential Cause (s)	Suggestion (s)
No amplification product in test or control samples	Amplification reaction was incorrectly set up	Optimize the proper reaction set up
	Improper storage has led to loss of activity of PCR reagents	Replace all components with fresh reagents
	Primers are not optimal and did not anneal	Redesign primers
Amplification worked in the control samples, but not in test samples	Digestion was incomplete	Extend digestion time up to 30 minutes at 55°C
	Samples were stored too long and genomic DNA degraded	Collect fresh mouse tail samples for genomic DNA extraction
	The lysis solution was mixed with PCR mixture for too long	PCR reactions should be initiated within 5 hours after template is added, and stored at 4°C in the mean time
	The quantity of the amplification product was not sufficient	Increase the number of PCR cycles to 35-40 to yield more amplification product
Non-specific amplification product (s)	Annealing temperature was too low	Increase the annealing temperature
	The number of PCR cycles was too high	Decrease the number of cycles to 30-35
	Primer concentration was too high	Decrease primer concentration
	Template concentration was too high	Dilute template in purified H₂O or TE buffer

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