• Nuclei lysis buffer
  0.32 M Sucrose
  0.01 M Tris pH 7.6
  0.005 M MgCl₂
  1% Triton X-I 00
  
  Dissolve 109.6 gm of sucrose in 400 ml of deionized water. Then add 10 ml of 1 M Tris pH 7.6, 5 ml of 1 M MgCl₂, and 10 ml of Triton X-100. Bring the volume to 1 L. Sterilize by autoclaving. Sucrose is sensitive to heat so autoclave for a minimum amount of time.
  
  
• Proteinase K buffer
  0.025 M EDTA pH 7.6
  0.15 M NaCl
  0.04 M Tris pH 7.4
  
  Mix 48 ml of 0.5 M EDTA pH 7.6, 30 ml of 5 M NaCl, and 40 ml of 1 M Tris pH 7.4 and bring up to 500 ml. Sterilize by autoclaving.
  
• Proteinase K stock
  10 mg/ml
  
• RNAse A stock
  10 mg/ml

Nuclei Preparation 
(Genomic DNA can be isolated from either purified nuclei or whole cells.  This protocol is for preparation of DNA from nuclli)

• Solid Tissue
  
  1. Grind 0.2 to 0.4 gm of tissue in 4.5 ml saline supplemented with 500 ul of 10X TE (use 15 ml polypropylene tube).|Spin 250g for 5 minutes (1000 rpm on the Beckman table top GPR centrifuge).
     
  2. Decant supernate.
  3. Lyse the cells once by adding 10 ml of cold nuclei lysis buffer and mix well.
     
  4. Spin 1500g for 15 minutes (2500 rpm on Beckman table top GPR centrifuge. DO NOT spin any harder than this. If the nuclei are too packed, they cannot be resuspended well and the resulting DNA will be clumpy).
     
  5. Decant supernate and invert tube on clean paper towel to drain the lysis buffer.
     
• Blood
  There are two ways of doing this: lyse the cells directly or separate the WBC before lysis.
  
  
  • Whole Blood Lysis
    The advantage of this method is that no cell separation is required, but the cells need to be lysed twice for complete lysis. Usually high gravity centrifugation is required after the first lysis to pellet the nuclei.
    
    1. Mix 1 ml of blood with 10 µl of cold nuclei lysis buffer in 15 ml polypropylene tube.
       
    2. Spin 1500g for 15 minutes (2500 rpm on the Beckman table top GPR centrifuge. DO NOT spin any harder than this. If the nuclei are too packed, they can not be resuspended well and the resulting DNA will be clumpy).
       
    3. Decant supernate.
       
    4. Repeat lysis and spin 1500g for 15 minutes (at this step, spinning in the Beckman table top GPR centrifuge is sufficient. DO NOT spin any harder this. If the nuclei are too packed, they cannot be resuspended well and the DNA will be clumpy).
       
    5. Decant supernate and invert tube on clean paper towel to drain the lysis buffer.
       
       
  • WBC Lysis
    The advantage of this method is that cells need to be lysed only once,no high gravity centrifugation is required and the DNA may be somewhat cleaner.
    
    1. Spin 1 ml of blood in 4 ml polypropylene tube at 1500g for 25 minutes (2500 rpm on the Beckman table top GPR centrifuge). The WBC should be separated as a white layer buffy coat between the top plasma layer and the bottom RBC layer.
       
    2. Harvest the WBC layer into 15 ml polypropylene tube (try to minimally take up RBC but some RBC contamination will not hurt).
       
    3. Add 10 ml of nuclei lysis buffer.
    4. Spin 1500 g for 15 minutes (2500 rpm on the Beckman table top GPR centrifuge. DO NOT spin any harder than this. If the nuclei are too packed, they cannot be resuspended well and the DNA will be clumpy).
       
    5. Decant supernate and invert tube on clean paper towel to drain the lysis buffer.
       
       
• Tissue Culture
  
  1. Lyse 10-20 x l0⁶ cells once with 10 ml of nuclei lysis buffer in 15 ml polypropylene tubes.
  2. Spin 1500g for 15 minutes (2500 rpm on the Beckman table top GPR centrifuge). DO NOT spin any harder than this. If the nuclei are too packed, they cannot be resuspended well and the DNA will be clumpy).
     
  3. Decant supernate and invert tube on clean paper towel to drain the lysis buffer.

1. Clean the nuclei lysis buffer from the side wall of the tube as much as possible using sterile cotton swab.
   
2. Take up 300 µl of proteinase K buffer using P1000 pipetman and a truncated tip.
   
3. Without dispensing the buffer, stir the nuclei pellet with the pipet tip and dispense the buffer after the pellet is deaggregated. Transfer the nuclei to a 1.5 ml Eppendorf tube.
   
4. Rinse the 15 ml tube with another 300 ul of nuclei lysis buffer and pool the nuclei.
5. Add 12 µl of 10 mg/ml proteinase K stock (add proteinase K first and mix well before adding SDS. The DNA becomes viscous when SDS is added and will be more difficult to mix with proteinase K if SDS is added before proteinase K).
   
6. Add 30 µl of 10% SDS.
   
7. Mix gently and incubate at 37°C overnight.
   
8. The next day, add 4.5 µl of 10 mg/ml RNAse A stock (boil the RNAse A for 10 minutes before use) incubate at 37°C for 1-4 hours.

1. Add 600 ul of Phenol to the DNA prep.
   
2. Gently invert tube up and down for 2 minutes.
   
3. Spin 5 minutes in table Eppendorf centrifuge at room temperature.
   
4. Transfer the upper phase (DNA layer) to a new Eppendorf tube (DNA should be viscous so use large bore pipet tip. DNA will not be separated well from the phenol and it is OK to carry over some phenol to the new tube).
   
5. Repeat the phenol extraction.
   
6. Add 600 µl of 1:1 mixture Phenol:Sevag.
   
7. Gently invert tube up and down for 2 minutes.
   
8. Spin 5 minutes in table Eppendorf centrifuge at room temperature.
   
9. Transfer the upper phase to new a Eppendorf tube (DNA should be well-separated from the organic phase).
   
10. Repeat the phenol:Sevag extraction.
    
11. Add 600 µl of Sevag.
    
12. Gently invert tube up and down for 2 minutes.
    
13. Spin 5 minutes in table Eppendorf centrifuge at room temperature.
    
14. Transfer the upper phase to a new Eppendorf tube.
    
15. Repeat the Sevag extraction.
    
16. Dialyze the DNA against a large volume of 1X TE at room temperature or in the cold with two changes of TE (use large diameter dialysis tubing because the DNA is viscous. Large diameter tubing allows easy retrieval of viscous DNA after the dialysis).