DNA Extraction protocol
(Procedure)
Method
Qiagen Easy DNA blood and Tissue Kit will be used.
1-Bring all reagents at room temperature
2-Prepare the reagents as mentioned in the handbook (done by the instructor)
3-Add 20 µl proteinase K to the bottom of a 1.5 ml micro-centrifuge tube.
4-Add 200 µl of the blood sample to a micro-centrifuge tube.
5-Add 200 µl Buffer AL to the sample
6-Mix by pulse- vortexing for 15 s.
7-Incubate at 56c for 10 min.
*briefly centrifuge the 1.5 ml microcentrefuge tube to remove drops from the inside of the lid
8-Add 200 µl ethanol (96-100%) to the sample
9-Mix by pulse- vortexing for 15 s.
*After mixing, briefly centrifuge 1.5 ml microcentrifuge tube to remove drops from the inside of the lid
10-Carefully apply the mixture to the mini spin column without wetting the rim
11-Close the cap and centrifuge at 8000 rpm for 1 min.
12-Place the Mini spin column in a clean 2 ml collection tube
13-Discard the tube containing the filtrate.
14-Carefully open the Mini spin column
15-Add 500 µl buffer AW1 without wetting the rim.
16-Close the cap
17-Centrifuge at 8000 rpm for 1 min
18-Place the Mini spin column in a clean 2 ml collection tube
19-Discard the collection tube containing the filtrate.
20-Carefully open the Mini spin column
21-Add 500 µl buffer AW2 without wetting the rim.
22-Close the cap
23-Centrifuge at 14,000 rpm for 3 min.
24-Place the Mini spin column in a clean 1.5 ml microcentrifuge tube
25-Discard the collection tube containing the filtrate.
26-Carefully open the Mini spin column
27-Add 200 µl buffer AE (or distilled water).
28-Incubate at room temperature for 5 min
29-Centrifuge at 8000 rpm.
30-Collect the filtrate in the tube and store it at 4 c until use in the PCR experiment next LAB.
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