This is a cached page for the URL (http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/Methods/DNAIsolation/phageQuickprep.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Lambda Phage DNA Quickprep

Zürich Online Bibliothek Uni ZH Vorlesungen Institute of Plant Biology Home Page U of Zürich Home Page   Schneitz Lab Home Page / Methods / DNA Work / Lambda Phage DNA Quickprep
 
 
Lambda Phage DNA Quickprep
Reference:  modified from Blattner et.al., 1978, Science 202:1279-1284 by Rick Garber and Markus Noll Last updated: 1/27/00 By: Kay Schneitz
     
 
  1. suspend a single plaque in 1 ml PSB
  2. adsorb 10 min at 37°C: 0.1 ml eluted phage*/0.1 ml MgCa/0.1 ml saturated K802 culture grown in NZY broth/0.2% maltose
  3. use 10 ml of this to inoculate 2.5 ml NZY, shake ovn at 37°C or until lysed
  4. cool to RT, then add 2 drops of CHCl3
  5. add 5 ml of 10 mg/ml DNase I (20 mg/ml final) and gently shake for 25 min
  6. divide lysate into two Eppendorf tubes, spin 5 min
  7. transfer 0.6 ml of each tube into a new tube prefilled with 200 ml of STE
  8. mix and incubate 15 min at 70°C
  9. cool to RT, add 150 ml 8 M KAc, mix and keep on ice for 15 min, spin for 15 min
  10. save 700 ml of clear supernatant, extract 1x with phenole/chloroform (vortex 5 sec)
  11. add 420 ml 2-propanol, mix and let sit at RT for 10 min
  12. spin for 8 min, very carefully remove supernatant (pellet does not stick to bottom of tube very well)
  13. wash pellet twice with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
  14. digest pellet in 50 ml each of 0.3 mg/ml panc. RNase in TE, pH 8.0 for 30 min at 37°C
  15. pool to 100 ml per sample, add 40 ml of 5 M NH4Ac and 200 ml 2-propanol
  16. keep 10 min at RT, spin 10 min
  17. remove supernatant, wash with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
  18. take up pellet in 20 ml 1x TE, usually 10 ml can be used for a restriction digest

 

Solutions:

 

STE:

1.5 % SDS, 0.3 M Tris (pH 9), 0.15 M EDTA

STE
 
10 % SDS 7.5 ml
2 M Tris pH 9 7.5 ml
0.5 M EDTA 15.0 ml
Total 50.0 ml
 

l-dil:

10 mM Tris-HCl pH7.5/10 mM MgSO4, autoclave before use

l-dil
 
2 M Tris-HCl pH 7.5 5.0 ml
1 M MgSO4 10.0 ml
H2O 985.0 ml
Total 1 l
     

MgCa:

10 mM MgCl2/10 mM CaCl2, autoclave before use

MgCa
 
1 M MgCl2 5.0 ml
1 M CaCl2 5.0 ml
H2O 990.0 ml
Total 1 l
 

PSB:

10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM MgCl2, 0.05 % gelatine; store over CHCl3

PSB
 
2 M Tris-HCl pH 7.5 1.0 ml
5 M NaCl 4.0 ml
1 M MgCl2 2.0 ml
0.5 % gelatine 20.0 ml
H20 173.0 ml
Total 200.0 ml
     

 

Remarks:

*: this value holds true for genomic phages (EMBL4, Lambda DASH), if using cDNA phages (lgt10/11, Lambda ZAP) take: 1 ml of eluted phage in each of 0.1 ml l-dil, MgCa and K802. This is a tricky protocol. However, when strictly adhering to the indicated treatment times and generally experimenting carefully it should work allright.

 

 
 Top
 

  © Copyright 1997-2000, Kay Schneitz. Last updated 1/21/00; 9:05:49 PM. Send comments to the webmeister.