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:6/27/2016)... , ... June 27, 2016 , ... ... their findings on what they believe could be a new and helpful biomarker ... new research. Click here to read it now. , Biomarkers are ...
(Date:6/27/2016)... ... 2016 , ... Newly created 4Sight Medical Solutions ... healthcare market. The company's primary focus is on new product introductions, to include ... are necessary to help companies efficiently bring their products to market. , The ...
(Date:6/27/2016)... 27, 2016  Liquid Biotech USA ... of a Sponsored Research Agreement with The University ... (CTCs) from cancer patients.  The funding will be ... correlate with clinical outcomes in cancer patients undergoing ... then be employed to support the design of ...
(Date:6/24/2016)... , June 24, 2016  Regular discussions on a range ... place between the two entities said Poloz. Speaking ... Ottawa , he pointed to the country,s inflation ... federal government. "In ... "Both institutions have common economic goals, why not sit down ...
Breaking Biology Technology:
(Date:5/20/2016)... -- VoiceIt is excited to announce its new marketing ... working together, VoiceIt and VoicePass will offer an ... slightly different approaches to voice biometrics, collaboration between ... Both companies ... "This marketing and technology partnership allows VoiceIt ...
(Date:4/28/2016)... BANGALORE, India , April 28, 2016 ... of Infosys (NYSE: INFY ), and Samsung SDS, ... partnership that will provide end customers with a more ... payment services.      (Logo: http://photos.prnewswire.com/prnh/20130122/589162 ) ... financial services, but it also plays a fundamental part in ...
(Date:4/15/2016)...  A new partnership announced today will help ... in a fraction of the time it takes ... life insurance policies to consumers without requiring inconvenient ... Diagnostics, rapid testing (A1C, Cotinine and HIV) and ... weight, pulse, BMI, and activity data) available at ...
Breaking Biology News(10 mins):