실험 Q&A Mol. Biol.-DNA > DNA Modification
gDNA 추출
레벨2 z크핫z (대학생)
안녕하세요 새내기 대학원생입니다. AccuPrep-Genomic-DNA-Kit 를 사용하여 Cell에서 gDNA를 추출하는 실험을 했습니다.
예전에도 Bric에 자문을 구한적이 있으나, 잠시 실험을 중단후 다시 했습니다. 그림에서 밴드가 깨지며 밑으로 backgound 가 나타나고 있습니다.
1. Centrifuge the cultured cells (104-106) for 5min at 300 x g. Discard the supernatant carefully without disturbing the pellet.
2. Resuspend the pellet in 200 μl of 1xPBS.
3. Add 20 μl of Proteinase K (see “Before you begin”) to the each tube.
4. Add 10 μl of RNase A (see “Before you begin”) to each tube and incubate the tubes for 2min at room temperature.
5. Go to step 3of “DNA Extraction from Whole Blood and Buffy Coat” in page 4 and continue the instructions accordingly.
6. Add 200 μl of GB Buffer to the sample and mix immediately by vortex mixer.
7. Incubate at 60℃ for 10 min.
8. Add 400 μl of absolute ethanol and mix well by pippetting.
9. Carefully transfer the lysate into the upper reservoir of the Binding column tube (fit in a collection tube) without wetting the rim.
10. Close the tube and centrifuge at 8,000 rpm for 1 min.
11. Discard the solution from the collection tube and reuse the collection tube.
12. Add 500 μl of WA1 Buffer without wetting the rim, close the tube, and centrifuge at 8,000 rpm for 1 min.
13. Discard the solution from the collection tube and reuse the collection tube.
14. Add 500 μl of W2 Buffer without wetting the rim, close the tube, and centrifuge at 8,000 rpm for 1 min.
15. Discard the solution from the collection tube and reuse the collection tube.
16. Centrifuge once more at 13,000 rpm for 1 min to remove ethanol completely, and check that there is no droplet clinging to the bottom of Binding column tube.
17. Transfer the Binding column tube to a new 1.5 ml tube for elution (supplied), add 50 - 200 μl of EA Buffer (or nuclease-free water) onto Binding column tube, and wait for at least 1 min at RT (15 - 25℃) until EA is completely absorbed into the glass fiber of Binding column tube.
18. Centrifuge at 8,000 rpm for 1 min to elute.
protocol 이며 Proteinase k 를 넣고 Ranase A를 넣고 37도씨에 5분 반응후 GB Buffer넣고 voltexing 대신에 inverting를 즉시 하였으며,
incubation 60℃에 10분 두고 absolute ethanol 를 mix 후 inverting을 하였습니다. 그리고 마지막엔 elution buffer를 50ul를 넣고 37℃에 5분간 두고
1% 전기영동으로 gDNA 10ul + 6 x dye 2ul를 넣고 Loading을 하였습니다.
제 손이 똥손인건 맞지만 몇번을 하는 실험인데... 이렇게 나오고 있습니다.
답답하시더라도 도와주시면 감사하겠습니다. ㅠㅠ
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