Table 2. Problem handling.

	Step	Problem	Possible reason	Solution
1	Reagents setup	We observed loss of PCR efficiency after multiple freezing-thawing cycles.	Primer degradation	To avoid degradation, aliquoted primer mixes should not be thawed more than 3–4 times. Small portions (50–100 μl) of primer mixes should be kept at -20°C.
2	Reaction setup	Uniformity	Reaction volume is small (5 μl). Preparation of single reaction that requires pipeting of volumes below 0.5 μl is very difficult.	We mix enough mastermix to fill whole 96 well plate and run multiple reactions at the same time.
3	Reaction setup	Uniformity	Pipetting errors caused by manual pipetting and small reaction volume.	We usually use an electronic pipette with the “dispense” mode.