Low Yield from Tissue Samples

Symptom

DNA recovery from tissue significantly lower than expected, particularly from muscle, brain, or metabolically active organs. Upper phase transfer yields less DNA than anticipated.

1. 1 Incomplete tissue homogenization preventing optimal lysis and nuclease inactivation
2. 2 Incomplete transfer of upper phase during protein separation; most DNA concentrated near protein phase interface
3. 3 For low-end input amounts: 45 min agitation time too long, causing DNA loss in low-content tissues (muscle, brain)
4. 4 Fresh tissue not processed immediately, allowing metabolically active organs to degrade DNA
5. 5 Tissue pieces too large for efficient homogenization and rapid Proteinase K access

1. 1 Use included pestle or rotor-stator homogenizer thoroughly; with rotor-stator, homogenize until all visible tissue pieces are gone
2. 2 Transfer as much of upper phase as possible during protein separation; DNA gradient concentrated closest to protein phase
3. 3 For input near lower range: reduce agitation time from 45 min to 15 min, finish incubation without agitation (increases yield 50–100% for low-DNA tissues)
4. 4 Process fresh tissue samples immediately, especially metabolically active organs (pancreas, intestine, kidney, liver)
5. 5 Work with smallest possible tissue pieces; homogenize into thinnest layer with pestle or until no visible pieces with rotor-stator