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DNA extraction from human blood by phenol chloroform method


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#1 meryn

meryn

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Posted 28 June 2011 - 11:17 PM

i had standardized the protocol for DNA extraction from human blood using the following protocol
Reagents:
1. Red Blood Cells Lysis Buffer
a. NH4CL : 8.29g
b. KHCO3 : 1g -made upto 1000ml with D/W/. pH adjusted to 7.4
c. Na2EDTA : 0.034g

2. 10% SDS
10g Sodium Dodecyl sulphate in 100ml of autoclaved double distilled water

3. 5M NaCl (50 ml)
14.62g of NaCl in 50ml of Double distilled water

4. Phenol : Cholorofom : Isoamylalcohol
25 : 24 : 1 (50ml)

5. 100% chilled absolute alcohol

6. 70% chilled Alcohol
----------------------------------------------------------------------------------------------------------------------------
1. 1ml of blood + 1ml of RBC lysis buffer. Centrifuged at 4,500rpm for 15min at room temperature
Repeated 3-4 times till creamy white pellet is obtained.

2. Pellet washed with 1ml of ice cold milli-Q water.
4,500 rpm for 5 min at room temperature.

3. Supernatant discard, pellet treated with 15ul of 10% SDS, 30ul of 5M NaCl, 500ul of ice cold Milli Q water and 150ul of Phenol-chloroform.
Centrifuged at 4,500rpm for 20min at room temperature.

4. 3 layers separate out. Superficial clear layer pippetted out into a fresh tube to which double the volume of ice cold ethanol is added
.
5. The DNA that spools out is picked up and washed with 70% alcohol twice and then dissolved in milli Q water once the alcohol has completely evaporated.

The DNA obtained was quantified using a nanodrop and the yeild as well as 260:280 ratio was good. I extracted DNA for 25samples using the same protocol.

But now not able to extract DNA at all. Nothing happens on mixing the aqueous layer with ice cold ethanol. No other step changed except that the speed was increased from 4,500rpm to 7,800 rpm.
Any suggestions/advice?




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