dna prep for eukaryotic cells (macrophages)?

Dr David E Barton dbarton at acer.gen.tcd.ie
Fri Jul 21 03:42:57 EST 1995



>>i'm looking for a commercial kit or a protocol to isolate
>>chromosomal dna from macrophages.
>>thanks for any info

We use the following prep for large-scale extractions,
but it can be scaled up or down as you please. It is an adaptaton
of the best commercial kit we have found (PureGene)

Procedure:

Add 3ml blood to 9ml cold RBC Lysis soln
Stand 15min, inverting occasionally
Spin 2,000g (speed not critical) 5-10min
You should see a white leukocuyte pellet now.
Pour off supernatant, leaving 100-200ul residual liquid
Vortex vigorously to resuspend
Add 3ml Cell Lysis soln - pipet up and down to lyse fully (we use disposable
pastettes)
Samples are stable like this for at least 18 months at room temp.
Add 1ml Protein Precipitation Soln
Vortex vigorously to mix
Centrifuge at 3,000g+ for 20min (Pellets proteins)
Pour the supernatant into a clean tube containing 3ml Isopropanol
Invert many times to form precipitate
Spin 3,000g 3min
Pour off supernatant
Add 3ml 70% Ethanol, invert a few times
spin 3,000g 1min
Drain tube on clean absorbent paper and allow to air-dry about 15min.
Resuspend in 250ul TE (This can be done by shaking the tubes overnight
in an orbital or by pipetting up and down and/or heating to 650C.

We also found that 1 to 4 mls of blood can be loaded into   
this 3ml prep with no adjustment required. Going to 5ml sometimes gave a
poorer 260/280 ratio, but even then we haven't seen any functional problems
with this DNA.

RBC Lysis:      155 mM Ammonium Chloride                                       
                 10 mM Potassium Hydrogen Carbonate                            
                  1 mM EDTA                                                    
                                                                               
Cell Lysis:     25 mM EDTA                                                     
                 2% SDS                                                        
                                                                               
Protein Precipitation soln: 10 M Ammonium Acetate
                                          
Rehydration Solution (TE):	10mM Tris.Cl pH 8.0
					1mM EDTA 

This protocol was adapted by Teo Hsiang Ling in my Lab.
                                                                               
Best wishes, David.                                                            
                                                                               
 \|/ ____ \|/  | David Barton                                                  
  @~/ oo \~@   | National Centre for Medical Genetics                          
 /_( \__/ )_\  | Our Lady's Hospital for Sick Children                         
    \__U_/     | Crumlin, Dublin 12, Ireland.
    _/  \_     | Tel +353 1 455 0515 Fax 455 8873




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