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DNA extraction from yeast by Harjul method (Protocol)

شارك:

  1. prepare yeast broth media 
  2. inoculate with yeast 
  3. incubate at 37°C. overnight 
  4. take 1.5 ml of yeast ,centrifuge it at 14000 rpm from 1-5 mins 
  5. power (discard) supernatant 
  6. add 200 μl     from  Harju buffer then vortex 
  7. transfer it between (water bath at 95°C. for 2 mins and ice at -5°C.  for 2mins) 2 times 
  8. add phenol chloroform and invert tube 
  9. centrifuge at maximum speed (14000 rpm) for 3 mins to obtain layers of (protein-DNA-RNA)
  10. transfer aqueous phase into new eppendorf 
  11. add 400 μl    absolute cold (ethanol) or  use isopropyl alcohol for 10 mins at room temperature 
  12. centrifuge at max speed for 3 mins 
  13. washing with 500 μl    with 70% ethanol for 5 mins 
  14. Repeat step 12(centrifuge at max speed for 3 mins)
  15. add 25-50 μl   TE buffer (PH=8) 
  16. Breaking proteins by adding a protease (optional).
  17. Breaking RNA by adding an RNASE (optional).




COMPOSITION :

Harju buffer :

2% tritionx 100 
1% SDS
100 mM Nacl 
10 mM Tris cl (PH =8)
1 mM EDTA

  • function: 

  1. (PH =8) for denaturation for protein 
  1. ethanol and isopropyl alcohol for precipitation of DNA and aggregation (


The difference between isopropanol and ethanol is the solubility of DNA in each solvent 
DNA is less soluble in isopropanol so it will fall out of solution faster and at a lower concentration, but the downside is that the salt will too. With ethanol, the DNA needs to be at a higher concentration to flocculate but the salt tends to stay soluble, even at cold temperatures.
DNA falls out of solution in 35% isopropanol and 0.5M salt. Using ethanol, the final concentration needs to be around 75% with 0.5M salt. So for the typical precipitation protocol, isopropanol is added from between 0.7-1 volumes of sample and ethanol is added at 2-2.5 volumes of sample.)

    3.Phenol chloroform is  used to separate nucleic acid from proteins and lipids

(because the phenol chloroform mixture is immiscible with water. The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The proteins and hydrophobic lipids will partition into the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. The upper aqueous phase is pipetted off and care is taken to avoid pipetting any of the organic phase or material at the interface. This procedure is often performed multiple times to increase the purity of the DNA.

If the mixture is acidic, DNA will precipitate into the organic phase while RNA remains in the aqueous phase due to DNA being more readily neutralised than RNA.)
   4.Sodium Chloride (NaCl) helps to remove proteins that are bound to the DNA. It also helps to keep the proteins dissolved in the aqueous layer so they don't precipitate in the alcohol along with the DNA.

REFERENCES: 

THIS experiment is related to labs at Helwan university 
2. Amberg DC, Burke DJ, Strathern JN. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. 2005 Edition 2005.
3. Harju S, Fedosyuk H, Peterson KR. Rapid isolation of yeast genomic DNA: Bust n’ Grab. BMC biotechnology. 2004;4:8. [PMC free article] [PubMed]
4. Akada R, Murakane T, Nishizawa Y. DNA extraction method for screening yeast clones by PCR. BioTechniques. 2000;28:668–670. 672, 674. [PubMed]
5. Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. Journal of bacteriology. 1983;153:163–168. [PMC free article] [PubMed]


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