Navigation Links
Mouse Tail Genomic DNA Isolation Protocol(1)

Note: Genomic DNA is fragile. High molecular weight DNA is sheared easily by mechanical forces. Use suitable large-bore pipet tips or equipment when pipetting genomic DNA. Do not vortex solutions containing genomic DNA.
  1. Place a 1 cm tail sample into a 1.5 ml microcentrifuge tube; this may be stored at 20C. To minimize possible cross-contamination, do not mince the sample. Add 700 l Tail Buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS) to the sample.
  2. Add 35 l 10 mg/ml Proteinase K to the sample and mix briefly.
  3. Incubate at 5560C overnight with mixing. This step should result in the complete solubilization of the tail fragment. In the case of incomplete digestion, more Proteinase K can be added and the samples incubated for several more hours.
  4. Add 20 l 10 mg/ml RNase A (DNase-free) to the sample. Mix briefly and incubate at 37C for 12 hours.
  5. Transfer entire solution to a pre-spun (1500 x g for 12 minutes) PLG 2 ml Heavy tube.
  6. Add 0.5 ml Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the sample in the PLG 2 ml tube and mix well by repeated inversion. Do not vortex.
  7. Centrifuge at full speed (12,000 x g or greater) for 5 minutes in a microcentrifuge, then carefully transfer the resultant aqueous phase to a fresh pre-spun PLG 2 ml Heavy tube.
  8. Add 0.5 ml Chloroform-Isoamyl Alcohol (CI, 24:1) to the sample in the PLG 2 ml tube and mix well by repeated inversion. Do not vortex.
  9. Centrifuge at full speed (12,000 x g or greater) for 5 minutes in a microcentrifuge, the n carefully transfer resultant aqueous phase to a fresh microcentrifuge tube.
  10. Fill sample-containing tube with 100% Isopropanol and mix thoroughly by repeated inversion. Do not vortex. A visible DNA precipitate should form. Proceed immediately to step 11.
  11. Recover DNA precipitate by touching it to a heat-sealed glass micropipette tip or by lifting the DNA with a yellow pipette tip and partial suction from a pipettor. Transfer the DNA to a 1.5 ml microcentrifuge tube containing 70% Ethanol. If DNA is not stringy, pellet by a brief, low speed centrifugation.
  12. Wash DNA with the 70% Ethanol, then wash twice with 95% Ethanol.
  13. Allow DNA to partially dry and then either transfer the DNA to a microcentrifuge tube containing 400 l TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) or add 400 l TE to the DNA in the tube. Do not vortex or re-pipet to resuspend DNA.
  14. Resolubilize the DNA overnight (e.g. by rotation at 3060 rpm). Resolubilization may be facilitated by heating the sample at 50C.
References
  1. Herrmann, B.G. and Frischauf, A.M. (1987) Isolation of genomic DNA. Methods Enzymol. 152:180-183.
  2. Laws, G.M. and S.P. Adams. (1996) Measurement of 8-OHdG in DNA by HPLC/ECD: The importance of DNA purity. BioTechniques 20:36-38.
  3. Murphy, N.R. and Hellwig, R. J. (1996) Improved nucleic acid organic extraction through use of a unique gel barrier matrix. BioTechniques 21:934-939.
  4. Fil Randazzo, personal communication.

'"/>

Source:


Page: All 1 2

Related biology technology :

1. RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes
2. Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies
3. Mouse Tail Genomic DNA Isolation Protocol(1)
4. Amplification of Mouse cDNAs for Microarrays Using the Eppendorf MasterTaq Kit
5. Cenix-Designed siRNAs for 95% of Human and Mouse Genomes
6. RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control
7. Simultaneous Detection of 8 Cytokines in Mouse or Human Sera
8. A Novel Process for Gene Expression Profiling of Rat and Mouse Tissues from Formalin-Fixed Paraffin-Embedded Sections Using Microarrays
9. Fast and Easy Isolation of PCR-Ready Genomic DNA from Whole Blood
10. New Protocols for Isolating High- Molecular-Weight Genomic DNA
11. Easily Amplify Genomic DNA with Long-Distance PCR
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/18/2017)... Kurzlehrgang mit Fokus auf ... Poster legt metaproteomische Analyse des Darm-Mikrobioms bei Säuglingen dar   ... ... Yoav Peretz , Scientific Director bei ImmuneCarta, einen ... zum Nachweis intrazellulärer Zytokine bei adoptiven Zelltherapie-Studien im Rahmen ...
(Date:2/18/2017)... ... 2017 , ... A leader in science-based, age-fighting skincare products, ... is proud to introduce its Age-Defying Night Cream, NeriumAD™ Formula, Age-Defying Day Cream, ... Hong Kong. The luxury skincare products contain innovative ingredients that fight the signs ...
(Date:2/17/2017)... , February 17, 2017 ... "Biomarkers Market by Product (Consumables, Service), Type (Safety, ... (Diagnostics Development, Drug Discovery and Development, Disease-Risk) - ... market is projected to reach USD 53.34 Billion ... growing at a CAGR of 13.8% during the ...
(Date:2/17/2017)... Feb. 17, 2017  If only one in ... a mutation-conferring resistance to chemotherapy, thousands of cancer ... has focused on finding these mutations in ever-smaller ... circulating tumor DNA in blood — to guide ... Unfortunately, however, detecting these genetic anomalies ...
Breaking Biology Technology:
(Date:2/2/2017)... Feb. 1, 2017  Central to its deep ... advances worldwide, The Japan Prize Foundation today announced ... have pushed the envelope in their respective fields ... Three scientists are being recognized with the 2017 ... not only contribute to the advancement of science ...
(Date:1/26/2017)... 26, 2017  Crossmatch, a leading provider of security ... aimed at combatting fraud, waste and abuse in assistance ... the Action on Disaster Relief conference in ... UN agencies and foreign assistance organizations throughout ... abuse are a largely unacknowledged problem in the foreign ...
(Date:1/24/2017)...  It sounds simple and harmless—an electronic sensor ... signs and alerts parents on their smart phones ... drops. But pediatric experts argue that such devices ... evidence of medical benefits, especially to healthy babies. ... parents of healthy babies, promising peace of mind ...
Breaking Biology News(10 mins):