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:6/26/2017)... ... June 26, 2017 , ... Third Wave Bioactives, LLC announces the addition ... on leading new business development and ensuring quality customer experience. , Brett ... ingredient industry in technical, marketing and sales roles. “Brett’s background working with customers and ...
(Date:6/23/2017)... , ... June 23, 2017 , ... Ken Hanson, a ... of Physik Instrumente USA, have been selected as this year’s recipients of two top ... two have been invited along with other honorees to accept their awards at a ...
(Date:6/22/2017)... ... June 21, 2017 , ... Building on the success of the inaugural RAADfest ... the very latest developments in radical life extension. RAADfest combines cutting edge science presented ... empowerment of personal development, making it the largest most comprehensive and inclusive super longevity ...
(Date:6/22/2017)... ... ... The first human cell line HeLa, established in 1951, has entered cell ... human cell lines with HeLa cells were published. Until recently, cross-contamination and misidentification of ... associated with dramatic consequences for research. , In this educational webinar, which is ...
Breaking Biology Technology:
(Date:4/17/2017)... Florida , April 17, 2017 NXT-ID, ... technology company, announces the filing of its 2016 Annual Report on ... and Exchange Commission. ... on Form 10-K is available in the Investor Relations section of ... as on the SEC,s website at http://www.sec.gov . ...
(Date:4/11/2017)... N.Y. , April 11, 2017 ... fingerprints, but researchers at the New York University ... College of Engineering have found that partial similarities ... security systems used in mobile phones and other ... thought. The vulnerability lies in the ...
(Date:4/5/2017)... -- KEY FINDINGS The global market for ... of 25.76% during the forecast period of 2017-2025. The ... the growth of the stem cell market. ... INSIGHTS The global stem cell market is segmented on ... stem cell market of the product is segmented into ...
Breaking Biology News(10 mins):