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:6/23/2016)... /PRNewswire/ - FACIT has announced the creation of ... company, Propellon Therapeutics Inc. ("Propellon" or "the Company"), ... portfolio of first-in-class WDR5 inhibitors for the treatment ... represent an exciting class of therapies, possessing the ... cancer patients. Substantial advances have been achieved with ...
(Date:6/23/2016)... 23, 2016 Houston Methodist Willowbrook Hospital ... Sports Association to serve as their official health ... Methodist Willowbrook will provide sponsorship support, athletic training ... association coaches, volunteers, athletes and families. ... Sports Association and to bring Houston Methodist quality ...
(Date:6/23/2016)... -- The Prostate Cancer Foundation (PCF) is pleased to announce 24 new ... prostate cancer. Members of the Class of 2016 were selected from a pool ... Read More About the Class of 2016 PCF Young Investigators ... ... ...
(Date:6/23/2016)...   EpiBiome , a precision microbiome engineering company, ... financing from Silicon Valley Bank (SVB). The financing will ... its drug development efforts, as well as purchase additional ... has been an incredible strategic partner to us – ... would provide," said Dr. Aeron Tynes Hammack , ...
Breaking Biology Technology:
(Date:6/27/2016)... Research and Markets has announced the addition of the ... offering. The report forecasts the ... at a CAGR of 12.28% during the period 2016-2020. ... analysis with inputs from industry experts. The report covers the market ... also includes a discussion of the key vendors operating in this ...
(Date:6/22/2016)... -- The American College of Medical Genetics and Genomics was once ... one of the fastest-growing trade shows during the Fastest 50 ... Las Vegas . Winners ... each of the following categories: net square feet of paid ... The 2015 ACMG Annual Meeting was ranked 23 out of ...
(Date:6/22/2016)... 2016   Acuant , the leading ... has partnered with RightCrowd ® to ... Management, Self-Service Kiosks and Continuous Workforce Assurance. ... functional enhancements to existing physical access control ... with an automated ID verification and authentication ...
Breaking Biology News(10 mins):