Navigation Links
Recovery of DNA from LMP Agarose1

Recovery of DNA from LMP Agarose1

  1. Resolve DNA fragments on a Low Melting Point (LMP) agarose gel in 1x Tris-Acetate-EDTA (TAE) buffer. Tris-Borate-EDTA (TBE) is not recommended as TBE gels are much more difficult to solubilize.
  2. Stain gel with Ethidium Bromide, visualize with a longwave UV light, and carefully cut out the band(s) of interest with a sharp razor blade. Caution: Wear gloves when handling Ethidium Bromide and stained gels.
  3. Transfer slice to a pre-spun (12,000 x g for 2030 seconds), pre-weighed PLG 2 ml Light tube and determine the weight of the slice.
  4. Add a volume (in l) of TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) equivalent to 5x the weight (in mg) of the slice and melt the LMP agarose in the TE at 65C for 510 minutes.
  5. Mix well to ensure LMP agarose slice is fully dissolved, allow the dissolved sample to come to room temperature, and then add an equal volume of room temperature Tris-buffer saturated Phenol (pH 8.0) to the sample and mix until homogeneous. Do not vortex.
  6. Centrifuge at full speed (12,000 x g or greater in a microcentrifuge) for 2 minutes to separate the phases. Note: If the resulting aqueous phase still appears cloudy, the extraction should be repeated with room temperature Tris-buffer saturated Phenol (pH 8.0). >
  7. Recover aqueous phase to a fresh PLG 2 ml Light tube and extract with an equivalent volume of room temperature Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1). Do not vortex.
  8. Centrifuge as in step 6 above . Recover aqueous phase to a fresh PLG 2 ml Light tube, and extract with an equivalent volume of room temperature Chloroform-Isoamyl Alcohol (CI, 24:1).
  9. Centrifuge as in step 6 above and recover the aqueous phase to a suitably sized microcentrifuge tube.
  10. Add 0.25 volume of 10 M Ammonium Acetate and 2.5 volume of 100% Ethanol to the sample and mix well.
  11. Incubate at room temperature for 20 minutes, pellet by centrifugation, wash pellet two to three times with cold 70% Ethanol, air-dry pellet, and resuspend in a suitable buffer.
References
  1. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp.6.30-6.31.

'"/>

Source:


Page: All 1 2

Related biology technology :

1. Efficient Recovery of Ultrapure Plasmid DNA
2. Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... Diego, CA (PRWEB) , ... June 27, 2016 , ... ... solutions for clinical trials, announced today the Clinical Reach Virtual Patient Encounter ... their care circle with the physician and clinical trial team. , Using the CONSULT ...
(Date:6/27/2016)... ... June 27, 2016 , ... ... will join the faculty of the University of North Carolina Kenan-Flagler Business ... strategy and entrepreneurship at UNC Kenan-Flagler, with a focus on the school’s international ...
(Date:6/27/2016)... Ginkgo Bioworks , a leading organism design company ... as one of the World Economic Forum,s Technology ... companies. Ginkgo Bioworks is engineering biology to manufacture ... the nutrition, health and consumer goods sectors. The ... Fortune 500 companies to design microbes for their ...
(Date:6/24/2016)... June 24, 2016 Epic Sciences unveiled ... cancers susceptible to PARP inhibitors by targeting homologous ... (CTCs). The new test has already been incorporated ... multiple cancer types. Over 230 clinical ... response pathways, including PARP, ATM, ATR, DNA-PK and ...
Breaking Biology Technology:
(Date:6/22/2016)... , June 22, 2016   ... management and verification solutions, has partnered with ... software solutions for Visitor Management, Self-Service Kiosks ... provides products that add functional enhancements to ... provides corporations and venues with an automated ...
(Date:6/16/2016)... , June 16, 2016 ... is expected to reach USD 1.83 billion by ... View Research, Inc. Technological proliferation and increasing demand ... are expected to drive the market growth. ... The development of advanced multimodal techniques ...
(Date:6/7/2016)... , June 7, 2016  Syngrafii Inc. ... a business relationship that includes integrating Syngrafii,s patented ... branch project. This collaboration will result in greater ... the credit union, while maintaining existing document workflow ... http://photos.prnewswire.com/prnh/20160606/375871LOGO ...
Breaking Biology News(10 mins):