NOTES: This preparation can be stored at 4 C for months. We typically use 1 ul of the DNA prep in a 10-15 ul reaction volume. It does not matter if fly parts (wings, bristles, legs) are inadvertantly added to the PCR mixture. Product will typically start to appear after 24-25 cycles, but 28-30 cycles seems to give maximal yield. Increasing the number of flies does not seem to increase the signal significantly, probably due to increasing concentrations of inhibitors. There should be no problem scaling up the number of flies screened if the volume is increased proportionately. 85 C heat inactivation allows longer fragments to be amplified -- up to more than 9 kb with ³long PCR²
NOTES: It is helpful to tape a piece of waxed paper over the open micro plate while the PCR tubes are being set up. That way the DNA samples can be drawn from each well by poking the pipet tip through the waxed paper. This procedure reduces the possibility of contamination and helps to keep track of which wells have been used.
NOTES: For the first attempt with a new insertion, we recommend using the following conditions: denature at 94 C for 45 sec., anneal at 60 C for 45 sec., extend at 72 C for 4 min; try 30 - 35 cycles. - The restriction enzyme appears to be the most critical component in this protocol. The enzyme must be specific under conditions of very low DNA concentration. Sau3A1, for example, is too promiscuous and digests at several sites in addition to its canonical restriction sequence. The protocol should work with other enzymes. The protocol has been used for a combined inverse/asymmetric PCR procedure to get DNA sequences flanking P element insertions.