I am currently facing difficulty in the DNA extraction because of the viscosity of the sputum samples. I am using the Chelex based extraction method which does yield in DNA but it is inefficient with the sputum samples treated with NaOH- NALC. I have come across some solutions such as cetylpyridinium chloride (CPC) and Dithiothreitol (DTT). Please guide me.
Many Thanks.
If NaOH-NALC treatment does not liquify your sample, you ca try this:
1- Do a pre-treatment with NaOH alone (final volume of 2 %), and then continue with your NaOH-NALC (fresh solution).
2- alternatively you can try the NaON 2%-Sodium Citrate 2.9%-NALC.
When using NALC, Avoid excessive vortexing since NALC is unstable in the presence of Oxygen.
Wich DNA extraction kit are you using ? if it's Epicentre (MasterPure), or other chelex-based method, you need to neutralize your sediment before procedding to culture, and /or DNA extraction. You can neutralize by using phosphate buffer (pH 6.8).
The principle of chelex method is a salting out method, the pH is critical for this method.
Finally how are you measuring DNA yield from sputum specimens ? it depends on your downstream testing methods: PCR, RT-PCR or sequencing.
Good luck
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