Hi Khawar,

I have tried and tested following two methods. You can give them a try. SDS method yields less DNA with greater purity than Proteinase K method and vice versa.

All the best.

• SDS Method: Resuspend 20-30mg cell pellet in 500ul SDS buffer (1%SDS, 3% NaCl) and vortex for 20 sec. Centrifuge at max speed for 1 min. Collect supernatant and add 500ul isopropanol, mix by inversion. Centrifuge at max speed for 1 min, remove supernatant. Add 500ul 70% ethanol (do not resuspend) and centrifuge again at max speed for 1 min. Remove ethanol and air dry the tube. Dissolve DNA in 50ul water. Use 2-5ul in 50ul PCR reaction
• Proteinase K Method: Resuspend 20-30 mg of cell pellet is in 100μl proteinase K "squishing buffer" (10 mM Tris-HCl, pH8; 1 mM EDTA; 25 mM NaCl; 200 μg/ml fresh Proteinase K) and incubate for 30 min at 37°C. Inactivate Proteinase K at 95°C for 5 minutes. Centrifuge at max speed for 1 min and use supernatant for PCR.

hi

use this article.

it is combination of sds and proteinase k.

i used it. it was excellent.

good luck

what is the actual question, do you have problem in DNA extraction or DNA concentration normalization ?

@abhijeet singh i am facing problem in normalization of DNA concentration.

How do you quantify the DNA concentration? Normalization is not difficult, you just have to use the formula C1V1=C2V2, normalize all of your samples to a certain concentration, so that you can use volume according to your need.

@abhijeet thank you for your reply. but that is not a point. i meant to say how can i optimize my procedure. i like every time when i extract the DNA , i need to change the Volume of dna solution from the mixture of the NAOH and TRIS HCl. for genotyping.