A Quick, No Frills Approach to Mouse Genotyping
小鼠基因型快捷分析法
发布: 2012年08月05日第2卷第15期 DOI: 10.21769/BioProtoc.244 浏览次数: 26797
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参见作者原研究论文
The authors used this protocol in:
Mar 2011
Abstract
Mice are extremely powerful mammalian genetic model organisms for basic and medical research, but managing a colony of transgenic mice is time consuming and expensive, many times requiring the help of dedicated technicians. Slow and laborious genotyping procedures add to the hassle. Outsourcing is costly and may not be as fast as desired, especially when setting up time sensitive experiments. Ultrafast genotyping protocols often require real-time PCR instruments and commercial reagents that may not be economical or practical. This protocol, adapted from methods suggested by The Jackson Laboratory, employs a minimalist approach that maximizes convenience by simplifying the tissue digestion/DNA extraction process and using a high-speed electrophoresis system for sample analysis. Genotype PCR results can be obtained in 3 h or less for as many samples as can fit in a PCR machine or can be efficiently handled by a user. Subsequent ethanol or chloroform purified DNA can be used in a standard PCR reaction to roughly identify a homozygous and a hemizygous mouse.
Materials and Reagents
- NaOH (NaOH pellets)
- Taq DNA Polymerase with ThermoPol buffer (New England Biolabs, catalog number: M0267X *, M0267L , or M0267S )
* Note: At 4,000U, ~800 μl of Taq serves several thousand PCR reactions. Buffer becomes a limiting reagent. ThermoPol Buffer recipe is available at NEB website. This buffer can be ordered separately from NEB (New England Biolabs, catalog number: B9004S ) - Primers, recommend to be 18-21 bp in length, have a melting temperature above 56 °C and around 58 °C, and produce amplicons of 150-600 bp.
- DNA loading buffer
Recommend Orange G (Sigma-Aldrich, catalog number: O3756 ) instead of bromophenol blue for loading dye - DNA ladder range for 100-800 bp range
Recommend 1 kb Plus DNA ladder (Life Technologies, catolog number: 10787-018 ) - Agarose
- EtBr (Sigma-Aldrich, catalog number: E8751 ) or SYBR Safe DNA Gel Stain (Life Technologies, catalog number: S33102 )
- NaOAc
- EtOH
- Phenol/Chloroform/Isoamyl alcohol (25: 24: 1) (Do not use acid phenol)
- 50 mM NaOH in dH2O (see Recipes)
- 10 mM dNTP Mix (see Recipes)
- 1x TAE buffer (see Recipes)
- dNTP set 100 mM each A, C, G, T (GE Life Sciences, catalog number: 28406552 ) (see Recipes)
- 0.3 M NaOAc in ddH2O (see Recipes)
Equipment
- PCR Thermal cycler (96 well capacity preferred)
- Centrifuges (mini for PCR tubes and microcentrifuge for 1.5 ml tubes)
- Liberty 1 buffer-less high speed gel system (Neuvitro, 6Mgel - SYS-LBT1)
- Liberty 1, 12-channel pipette compatible 13 teeth combs (Neuvitro, 6Mgel - CMS-1315)
- Multichannel Pipette 2-20 μl
- Repeat Pipettor 10-125 μl
- PCR tube with cap, 8 or 12 PCR strip tubes, or 96-well PCR plate
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Lopez, M. E. (2012). A Quick, No Frills Approach to Mouse Genotyping. Bio-protocol 2(15): e244. DOI: 10.21769/BioProtoc.244.
- Lopez, M. E., Klein, A. D., Dimbil, U. J. and Scott, M. P. (2011). Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder. J Neurosci 31(12): 4367-4378.
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分类
分子生物学 > DNA > 电泳
分子生物学 > DNA > 基因分型
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