I quantitated the DNA with a spectrophotometer but why did you find the template DNA insufficient?
A typical plasmid miniprep produces 5µg in 50µL. To save precious DNA from being wasted in what is viewed as destructive testing on the spectrophotometer, you remove 5µL from the 50µL plasmid prep. This amount is diluted with 500µL of water for the A260 measurement. Assuming all goes well, you get a reading of 0.02A260. Sounds good? I’m afraid not. What most operators don’t realize is that spectrophotometers can only read down to 0.05A260 reliably if they are properly blanked. Taking the above scenario, instead of 5µL, you used 20µL. This would have produced a reading of 0.05A260. Barely scraping past the accuracy of the spectrophotometer. Matters become worse since not all plasmid minipreps produce 5µg consistently. If you are dealing with a low copy plasmid, getting 1µg would be most fortunate. To be sure, assume that A260 readings from spectrophotometers to be suspicious. The above problems that face spectrophotometers are also found in the Nanodrop device since it is basically a sp
