It depends on the size of your target band. For medium and large targets between 300 and 1000 bp, we recommend control mix A (231 bp). For small targets (100-250 bp), control mix B should be used. If no internal control is desire, please use control mix C.

If you want to purify the PCR product and use it further, e.g. for DNA sequencing, we recommend to completely omit the control mix and to replace the missing volume in the PCR reaction by PCR water instead. Any gel loading buffer can be mixed with an aliquot of PCR product before gel electrophoresis.

The Snooplex lysis buffer is routinely used at GVG GM for DNA extraction from all types of mouse and rat sample material. It was found that the conditions originally recommended for swabs (30 minutes at 70°C) do not work for tail biopsies, for example. In a series of experiments it was investigated whether it is possible to define lysis conditions that are equally suitable for all samples. This is the case. We use incubation at 90°C for 60 minutes, with gentle shaking (500 rpm).

Important: Not too much sample material should be used for DNA extraction. 1-3 mm from a tail tip or 1 - 2 ear punches are completely sufficient to perform up to 50 PCR runs with 2 µl DNA extract each under optimized PCR conditions. The essential prerequisite for this is the use of the Snooplex PCR chemistry and the PCR conditions optimized for low DNA quantities (primer quantity).

The PCR reagents and control primers included in the kit are robust over a wide temperature range, so you can use your standard conditions. However, before routine use of the kit, it is recommended to check on an existing transgene-positive DNA sample whether the PCR works optimally. If not, the PCR parameters should be adjusted accordingly, e.g. different annealing temperature (+2 and -2°C), reduction of primer amount.

Important: For any PCR, there is always an optimal ratio between the number of DNA molecules (targets) and the amount of primers. The amount of DNA from swabs is considerably lower compared to tail biopsies. Therefore, the originally validated standard PCR conditions are logically suboptimal for low DNA amounts and should be adjusted. The two most important parameters to improve PCR conditions are the number of PCR cycles and the amount of PCR primers used. Therefore, use a DNA dilution series to test your standard conditions and adjust them if necessary. As a good orientation, how far you can dilute the DNA, serves the internal PCR control in the control mix A or B. As long as this band is still visible, there is a sufficient amount of DNA for a PCR. For this, increase the number of PCR cycles to 40. Usually, the amount of target-specific PCR primers added to the reaction can be reduced by 50% or even more. By the way, the optimal PCR conditions found for small amounts of DNA also work wonderfully for larger amounts of DNA.