I'm trying to extract DNA from nasal swabs using the TeNP40 method. This was the preferred method of extraction as a previous employee had used it before and we have to keep things consistent.
The problem is that we can't seem to get enough DNA out of the nasal swabs. Our protocol for TeNP40:
1. Add 400uL of 10mM Tris pH 8.0, 0.1mM EDTA, and 0.1% NP40 to your sample (usually swabs, or samples without any other liquid).
2. Add ~100uL of beads.
3. Boil at 95'C for 10 minutes.
4. Bead beat for 60 sec
5. Centrifuge at 10,000xg for 1 minute.
6. Transfer supernatant (avoiding beads and any gunk) to new clean tube
7. Add supernatant to a zymo column to clean and concentrate DNA
We quantified DNA using qPCR and the results from the qPCR and are around 35-38 Cts, which isn't a lot of DNA. The failure of some samples to amplify was likely due to insufficient biomass.
So I was wondering if we can optimize our protocols (such as bead beating longer or swab the patient harder or something, maybe specific pH levels...etc) using the same extraction method to increase biomass of DNA?
Thank you for your suggestions! Anything helps!
Here is a method I use in the lab:
1. Add 100-300uL of DNA SuperLyser* to the sample [samples including cultured cell, plant, virus, blood (dry or fresh), tissue, swabs and body fluid].
2. Incubate at room temperature for 30 min or heat for 20 min at 80-90 0C for tissue samples, Saliva, swabs, and dry blood.
3. Centrifuge at 12,000xg for 1 minute.
4. Transfer supernatant to a clean tube
5. Add 2 to 3 volume of BP buffer [5 M guanidine hydrochloride (Gu-HCL), 30% isopropanol] to the supernatant.
6. Mix well and load it to Enzymax DNA Tini spin column (designed for trace amount of DNA/RNA recovery and concentration) (http://www.enzymax.net/columns/tini_spin_DNA_column.htm)
7. Spin and discard the flow-through
8. Wash with 80% ethanol and elute the DNA in 5-20ul ddH2O
Note: SuperLyser* is a cocktail that specially formulated for nucleic acid isolation from various types of samples. Email for information at [email protected]
Hope this helps. Good luck!
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