- Minimize freeze-thaw cycles (3 max) on dNTP stock solution. 10 ul working aliquots and 50 / 100 ul storage solutions worked well for me.
- Volume of the reaction solution is another variable that should be considered. Upon optimization, I was able to successfully amplify templates with as much as 200 ul per PCR tube.
- Adding water first and reaction buffer second before the other reagents increased reproducibility (this is probably useful for any reaction).
- dNTPs will eventually settle out of solution. Always mix your dNTPs.