Hi Bhairavi, I am assuming you are looking for free DNA or cells from the baby?
For free DNA or for cells in the body fluid, treat it like blood- I would try UC Blood Spin DNA Kit from MO BIO Laboratories. http://www.mobio.com/blood-dna-isolation/ultraclean-bloodspin-dna-isolation-kit-1.html
You can get a free sample of the kit to test it.
Hi Bhairavi,
Here is the answer from our Technical Support Person at MO BIO:
I'm not sure what volume they would be starting with. However, I'd suggest that they centrifuge the liquid at 10,000 x g for 2 minutes to pellet the cells. They would then remove the liquid and start with this cell pellet.
If there are low levels of inhibitors and they want microbial DNA they could use the UltraClean Microbial kit. If the liquid is contaminated by blood, meconium etc. they could use the BiOstic Bacteremia kit starting from the cell pellet. This would remove inhibitors.
If they are looking for human DNA from cells and there are low levels of inhibitors, I'd suggest the UltraClean Tissue & Cells Isolation kit starting with the cell pellet, following the protocol for cultured cells.
If they want human cells and the fluid is contaminated then they could again use the BiOstic Bacteremia kit. But they would not use the bead tube. Instead they would start by resuspending the cell pellet in Solution CB1, vortex for 10 seconds to mix and place at 70 C for 15 minutes with 10 ul of Pro K. The rest of the protocol would be as usual.
I hope it helps.
Again we have samples of all the kits mentioned above if you feel like testing it.
Dear Bhairavi
Hello,
Here is a good protocol:
1. Divide the sample of amniotic fluid into labelled 2 mL eppendorf tubes (the number of tubes will depend on the volume of sample received). Spin in microcentrifuge at 13,000 rpm for 10 min. alternatively, centrifuge the cells in a larger tube, remove supernatant and transfer entire pellet to single eppendorf using PBS and then centrifuge eppendorf as above.
2. Remove supernatant.
3. Add 375 uL salting out buffer, 6.25 uL 20% SDS and 125 uL proteinase K (5 mg/mL)
4. Incubate Over night in 37C water bath or 30 - 60 min at 65C (flicking to resuspend constantly) to digest protein.
5. Label 3 X 1.5 mL microfuge tubes
6. To the digested sample add 400 uL phenol, invert and spin at 10,000 rpm in microcentrifuge for 5 min.
7. Transfer aqueous layer to another tube and add 400 uL 1:1 phenol:chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
8. Transfer aqueous layer to a new tube and add 400 uL chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
9. Transfer aqueous layer to a new microfuge tube and add 1/10 volume 3 M NaAc, pH 5.2 (40 uL) and 2.5 volumes of 100% ethanol (1 mL). Invert tube gently to precipitate DNA.
10. Spin at 10,000 rpm for 5 min and pour off ethanol.
11. Rinse the pellet once with 70% ethanol, centrifuge 10,000 rpm for 5 min. (Do not include this step if the pellet is very small)
12. Dry DNA pellet in laminar flow cabinet and resuspend in 20 - 50 uL of sterile water depending on size of pellet.
Salting out Lysis Buffer
1M Tris pH.7.5 10 mL
5M NaCl 80 mL
0.5 M Na2 EDTA 4 mL
Add dH2O to 1lt. and mix reagents with magnetic stirrer for short while. Label and date. Autoclave. Store at RT.
Good luck,
Hadi
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