Hi,
I need to send my samples for sequencing before that i need to purify my samples. can anyone please tell me how do i purify my samples without using a DNA purification kit?
Hi,
Usually, I performed the DNA purification through the electrophoresis as the following:
1. Cut the bands from the gel using Sharp blade under the UV.
2. Store the bands into cellulose bag filled with 0.5 ~1 μl 1 × TAE buffer.
3. Arrange the bands horizontally instead of cathode and anode for 5 min then change the cable for 5 sec.
4. Correct the solution by pipetting up and down.
5. Transferee the solution to 2 ml tube.
6. Centrifuge 10,000 g – 10 min/ 4° C.
7. Transfer 600 μl supernatant to 5 ml tube.
8. Add 75 μl EDTA (100 mM pH 8.0).
9. Add 57 μl NaAc (30 M pH 5.5).
10. Add 20 μl glycogen solutions (20 mg/ml) [you can reduce the amount to be half].
11. Complete the reaction up to 3 ~ 3.5 ml by absolute Ethanol 99.5 ~ 100% - Vortex.
12. Leave on ice (or at 4° C, or at room temperature) for 15 min.
13. Centrifuge 200,000 g – 35 min/ 4° C.
14. Discard the supernatant carefully.
15. Washing the pellets using 500 μl Ethanol 70%- Vortex thoroughly.
16. Centrifuge 200,000 g – 10 min/ 4° C.
17. Discard the supernatant carefully.
18. Dry the pellets in the speed-vac for 5 min, or air dry.
19. Add 30 μl 1/10 TE buffer.
20. Storage overnight in the fridge.
21. Measures the concentration by Nanodrop.
Hi Pooja,
I guess you need the old molecular biology techniques! Not too difficult, I give you two possibilities, depending on your equipment and consumables.
1) Quickest, but with low yield. Take a 1.5 ml eppi and with a needle 22-24 G make a hole directly in the bottom. Then fill the conical part of the eppi with some glass or plastic wool. Run your PCR on an agarose gel, cut out the band corresponding to your fragment (if you can avoid using 260 nm UV light, best is to use a 320 nm UV light) and cut it in small pieces. Place the pieces on the wool in your eppi, insert the eppi into another eppi (without any holes!) and spin at max. speed for 2-3 min. The collected liquid will contain your PCR fragment, with a yield of maybe 50%. There will be tiny fragments of agarose also and the Ethidium Bromide or similar DNA dye, so you will have to extract with phenol/chloroform pH 7.4, add 0.3 M NaOAc and precipitate in 2.5 vol. of EtOH.
2) Second possibility, if you have dialysis tubes (best the MW 4000 Da cut-off to purify also small DNA fragments), insert the agarose piece containing the band corresponding to your fragment in a short piece on this tube and close it on the sides with plastic clips. Then put it back in the gel running chamber, in the TE buffer, with the clips to the sides of the chamber (so that the long side of the tube and the agarose piece face the +/- poles) and run like a gel for 5-10 min: this will drag the DNA out of the agarose and against the wall of the tube, you can check when the DNA is all out with a UV transilluminator (again, use 320 nm if you can), you'll see a bright line along the wall of the dialysis tube and a dark agarose. Then take the tube gently, open one clip holding the tube vertical, cut the dialysis tube to open it and with a P200 Gilson aspirate the liquid in the tube washing carefully the side where the DNA accumulated. Transfer in eppi and, as above, extract with phenol/chloroform and precipitate.
As I mentioned, the yield of these methods is not high, so be sure you have enough of your specific band to start with, in case pool two or three PCR reaction and run them in large well (if you don't have combs with large wells, just make a large well from combs with small wells by fusing them with a piece of autoclave tape).
Good luck.
Mr. Pietro Pilo Boyl has explained the process. But its alow yield process.
Thank you
I use the freeze and squeeze tech.
As above in#1, but once i have the pcr dna in the gel cut out i place it into a 1.5 ml centrifuge tube, place in -80for a few minutes to freeze then centrifuge on high speed for a minute, when done there will be a smashed pelleded gel with liquid supernatant with your pcr dna.
Careful not to uv to long as could damage your dna when removing from gel.
Once i saw aresearcher cut a small hole just below the pcr product then run the gel until the band entered the hole and just pipet out.
Best of luck
Hi,
Usually, I performed the DNA purification through the electrophoresis as the following:
1. Cut the bands from the gel using Sharp blade under the UV.
2. Store the bands into cellulose bag filled with 0.5 ~1 μl 1 × TAE buffer.
3. Arrange the bands horizontally instead of cathode and anode for 5 min then change the cable for 5 sec.
4. Correct the solution by pipetting up and down.
5. Transferee the solution to 2 ml tube.
6. Centrifuge 10,000 g – 10 min/ 4° C.
7. Transfer 600 μl supernatant to 5 ml tube.
8. Add 75 μl EDTA (100 mM pH 8.0).
9. Add 57 μl NaAc (30 M pH 5.5).
10. Add 20 μl glycogen solutions (20 mg/ml) [you can reduce the amount to be half].
11. Complete the reaction up to 3 ~ 3.5 ml by absolute Ethanol 99.5 ~ 100% - Vortex.
12. Leave on ice (or at 4° C, or at room temperature) for 15 min.
13. Centrifuge 200,000 g – 35 min/ 4° C.
14. Discard the supernatant carefully.
15. Washing the pellets using 500 μl Ethanol 70%- Vortex thoroughly.
16. Centrifuge 200,000 g – 10 min/ 4° C.
17. Discard the supernatant carefully.
18. Dry the pellets in the speed-vac for 5 min, or air dry.
19. Add 30 μl 1/10 TE buffer.
20. Storage overnight in the fridge.
21. Measures the concentration by Nanodrop.
you wont get any standard reference for this in house protocol. from this you have to develop your own for your lab.. we also use freeze and squeezing protocol but before freezing we melt the agarose gel at 85 degree then freeze at -50 degree and then squeezing by centrifugation at high speed at 4 degree
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