Navigation Links
Lambda DNA Extraction Protocol


This method produces good quality LambdaDNA with yields of 1520 g lgt10 DNA per 450 l of supernatant processed. PLG tubes also can be used in conjunction with other LambdaDNA purification methods.(1)
  1. Grow a 10 ml stock of suitable plating bacteria in L-broth plus Mg2+ (1% Tryptone, 0.5% yeast extract, 0.5% NaCl, 1 mM MgSO4, pH 7.5) overnight at 37C.
  2. Dilute lambda phage stock to l05 pfu per 0.1 ml in SM buffer (0.1 M NaCI, 8 mM MgSO4, 50 mM Tris-Cl, pH 7.5, 0.01% Gelatin), then mix 0.1 ml of the lambda dilution with 0.1 ml plating bacteria in a sterile 13 x 100 mm culture tube and allow phage to adsorb to bacteria for 20 minutes at 37C.
  3. Add 2.5 ml melted top agarose (L-broth plus Mg2+ and 0.6% agarose, 4550C) to the tube, mix well and pour onto a 100 mm L-broth plus Mg2+ plate. Allow top agarose to harden and incubate at 37C for 8 hours. Plaques should be evident at this time.
  4. Chill plate for 15 minutes at 4C, add 3 ml lamda diluent (10 mM Tris-CI, pH 7.5, 8 mM MgSO4) to the plate and incubate overnight at 4C with gentle rocking.
  5. Collect phage-containing lambda diluent to a centrifuge tube and centrifuge at 4,000 x g for 10 minutes, 4C, to pellet debris.
  6. Transfer 450 l of the resultant supernatant to a pre-spun (1500 x g for 12 minutes) PLG 15 ml tube (Light or Heavy), add 4.5 l 1 mg/ml DNase I and 2.0 l 12.5 mg/ml RNase, mix and incubate 30 minutes at 37C.
  7. Add 11.5 l 20% SDS and 4.5 l 10 mg/ml Proteinase K to the sample, mix, and incubate 30 minutes at 37C.
  8. Extract sample with 500 l Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) and centrifuge for 5 minutes at 1500 x g to separate the phases.
  9. Repeat step 8 in the same PLG 15 ml tube.
  10. Using the same PLG 15 ml tube again, extract once with 500 l Chloroform-Isoamyl Alcohol (CI, 24:1) and centrifuge for 5 minutes at 1500 x g to separate the phases.
  11. Transfer final aqueous phase to a 1.5 ml microcentrifuge tube, add 45 l 3 M Sodium Acetate, pH 5.2 and 500 l 100% Isopropanol, mix and incubate at room temperature for 15 minutes.
  12. Centrifuge 20 minutes at 12,000 x g or more, discard the supernatant and wash pellet two times with 70% Ethanol prior to drying.
  13. Resuspend the DNA in 2050 l TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0).
References
  1. Current Protocols in Molecular Biology, Volume I. (1989) Ausubel, F.M. et al. (eds.). John Wiley and Sons, New York, NY pp.1.12.1 - 1.13.10.

'"/>

Source:


Page: All 1 2

Related biology technology :

1. A New Lambda Vector for Mammalian Expression
2. Highly Active and Removable AffinityTagged Lambda Phosphatase
3. Lambda DNA Extraction Protocol
4. M13/Phagemid DNA Extraction Protocol
5. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
6. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
7. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
8. M13/Phagemid DNA Extraction Protocol
9. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
10. S.E.T.S. Swab Extraction Tube System
11. InstaGene Matrix: Prepare DNA Templates for PCR With No Phenol/Chloroform Extractions and No Deproteinization Steps, Rev C
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/24/2016)... ... May 24, 2016 , ... Media Cybernetics, ... new Media Cybernetics corporate branding reflects a results-driven revitalization for a company with ... The re-branding components include a crisp, refreshed logo and a new web presence. ...
(Date:5/23/2016)... WARSAW, Ind. , May 23, 2016 Zimmer ... in musculoskeletal healthcare, today announced that its Board of Directors ... stockholders for the second quarter of 2016. ... on or about July 29, 2016 to stockholders of record ... Future declarations of dividends are subject to approval of the ...
(Date:5/23/2016)... ... May 23, 2016 , ... RoviSys, a leading independent provider ... Ohio, has broken ground on a new building in Holly Springs, NC. With ... new location solidifies a commitment to business in the region. The new facility ...
(Date:5/20/2016)... Diego, CA (PRWEB) , ... May 20, 2016 , ... ... that 10 of its most experienced veterinary clients have treated over 100 of their ... cutting edge technology to provide the highest level of care for their patients. ...
Breaking Biology Technology:
(Date:3/11/2016)... , March 11, 2016 http://www.apimages.com ) - ... reference: Picture is available at AP Images ( http://www.apimages.com ) - ... will be used to produce the new refugee identity cards. DERMALOG ... innovations, at CeBIT in Hanover next week.   ... DERMALOG will be used to produce the new refugee identity cards. ...
(Date:3/10/2016)... , March 10, 2016   Unisys Corporation ... and Border Protection (CBP) is testing its biometric identity ... San Diego to help identify certain non-U.S. citizens ... . The test, designed to help determine the efficiency and ... began in February and will run until May 2016. ...
(Date:3/9/2016)... , March 9, 2016  Crossmatch ® , ... enrollment solutions, today announced the addition of smart ... Altus multi-factor authentication platform. New contextual and ... to step-up security where it,s needed most — ... Washington, DC . --> ...
Breaking Biology News(10 mins):