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:6/20/2017)... ... June 20, 2017 , ... Do More with OHAUS , With the launch ... supplier in the weighing industry, to extending its expertise across the entire laboratory to ... hybridizations and more, allowing for its customers to 'Do More' in the ...
(Date:6/19/2017)... ... June 19, 2017 , ... EDETEK, Inc., ... today that it is launching two new additions of its award-winning cloud-based platform ... capabilities at the DIA 2017 Annual Meeting in Chicago, IL, June 19-22, 2017. ...
(Date:6/16/2017)... Massachusetts (PRWEB) , ... June 16, 2017 , ... ... medical device compliance and commercialization, has just announced two more sessions of its ... the series will focus on the world of online templates for design control ...
(Date:6/15/2017)... Colorado (PRWEB) , ... June 15, 2017 , ... ... the synthetic auxin herbicides give farmers new options for managing Palmer amaranth and ... of America (WSSA) say special precautions are necessary. Auxin herbicides are known to ...
Breaking Biology Technology:
(Date:3/22/2017)... , March 21, 2017 Optimove ... used by retailers such as 1-800-Flowers and AdoreMe, ... — Product Recommendations and Replenishment. Using Optimove,s machine learning ... personalized product and replenishment recommendations to their customers ... on predictions of customer intent drawn from a ...
(Date:3/20/2017)... HANOVER, Germany , March 20, 2017 At ... Hamburg -based biometrics manufacturer DERMALOG. The Chancellor came to the ... Japan is this year,s CeBIT partner country. At the largest ... important biometrics in use: fingerprint, face and iris recognition as well as ... ...
(Date:3/16/2017)... Germany , March 16, 2017 CeBIT 2017 - Against identity ... Continue Reading ... Used combined in one project, multi-biometric solutions provide ... Used ... Systems) ...
Breaking Biology News(10 mins):