Sputum should be collected into a sterile sputum collection cup, vial or tube.

Simply remix and centrifuge again. After centrifuging, decant the supernatant to a fresh tube.

Adding less volume may reduce DNA yields. Adding more may not affect the DNA yields except when performing the DNA elution step. Eluting DNA in higher volumes of elution buffer will result in diluting your DNA.

Yes, a second elution can be performed using a smaller volume of elution buffer B (50 µL). Please perform second elution in a new elution tube and ensure the concentrations before combining the elutions. Combining elutions can dilute the first elution if the second elution's concentration is too low.

Some sputum samples contain very little DNA. This will vary from individual to individual based on a number of variables. In order to increase the DNA yield it may be necessary to isolate DNA from a larger volume of sputum sample or isolate from several samples and then pool the DNA eluted from the two samples.

If a different elution buffer was used from the one provided in the kit, the buffer should be checked for components that may interfere with the application. Common components known to interfere with downstream assays include high salts, EDTA, detergents and other denaturants. Check the compatibility of the elution buffer with the intended use.

Let the elution stand vertically for 10 minutes to precipitate the resin and then use the clear elution supernatant for downstream applications. (Alternatively, centrifuge at low speed (200 x g for 1 minute.) This will neither decrease the DNA yield nor interfere with any downstream application.