on a [Na+] of 50 mM Controls Positive control: DNA from reference material (e.g. RRS 0.5% GM) Negative control: DNA from reference material (e.g. soybean 0% GM) No-template control (NTC): negative control of the Mastermix, in water is used instead of DNA Mastermix preparation The necessary reagents for a series of 10 samples (including positive/negative/no template controls) are mixed together according to the instructions given in Table 4. The following procedure applies to a sample containing 20 μL of HA-nos118-f/HA-nos118-r Mastermix and 5 μL of DNA solution. All solutions are stored on ice during the preparation of the Mastermix. Reagents Final concentration Volume for one Volume for 10 samples Sterile, deionised water 10.34 μL 103.4 μL 10x PCR buffer 1x 2.5 μL 25 μL 25 mM MgCl2 1.5 mM 1.5 μL 15 μL 4 mM dNTPs 0.64 mM 4 μL 40 μL 20 μM oligonucleotide 0.6 μM 0.75 μL 7.5 μL 20 μM oligonucleotide 0.6 μM 0.75 μL 7.5 μL 5 U/µL Taq DNA polymerase 0.8 U/rcn 0.16 μL 1.6 μL VOLUME 20 μL 200 μL Table 4. HA-nos118-f/HA-nos118-r Mastermix Prepare a 1.5 ml microcentrifuge tube Add the reagents following the order given in Table 4 Mix gently the HA-nos118-f/HA-nos118-r Mastermix by pipetting and centrifuge briefly Divide the Mastermix into aliquots of 20 μL in 0.2 ml PCR reaction tubes Add 5 μL of the DNA solution to the previous aliquots Shake gently and centrifuge briefly Place the PCR reaction tubes in the thermocycler PCR Program (HA-nos118-f/HA-nos118-r) Stage Temperature Time Initial Denaturation 95°C 10 min Denaturation 95°C 25 sec Annealing 62°C 30 sec Extension 72°C 45 sec Number of 50
on a [Na+] of 50 mM Controls Positive control: DNA from reference material (Bt-11 1% GM) Negative control: DNA from reference material 0% GM) No-template control (NTC): negative control of the Mastermix, in which water is used instead of DNA Mastermix preparation The necessary reagents for a series of 10 samples (including positive/negative/no template controls) are mixed together according to Table 6. The following procedure applies to a sample containing 20 μL of IVS2-2/PAT-B Mastermix and 5 μL of DNA solution. All solutions are stored in ice during the preparation of the Mastermix. Reagents concentration Volume for one Volume for 10 samples Sterile, deionised water 11.55 μL 115.5 μL 10x PCR buffer 1x 2.5 μL 25 μL 25 mM MgCl2 2 mM 2 μL 20 μL 4 mM dNTPs 0.4 mM 2.5 μL 25 μL 20 μM oligonucleotide IVS2-2 0.5 μM 0.63 μL 6.25 μL 20 μM oligonucleotide PAT-B 0.5 μM 0.63 μL 6.25 μL 5 U/µL Taq DNA polymerase 1 U/rcn 0,2 μL 2 μL TOTAL VOLUME 20 μL 200 μL Table 6. IVS2-2/PAT-B Mastermix Prepare a 1.5 ml microcentrifuge tube Add the reagents following the order given in Table 6 Mix gently the IVS2-2/PAT-B Mastermix by pipetting and centrifuge briefly Divide the Mastermix into aliquots of 20 μL in 0.2 ml PCR reaction tubes Add 5 μL of the DNA solution to the previous aliquots Shake gently and centrifuge briefly Place the PCR reaction tubes in the thermocycler PCR program (IVS2-2/PAT-B) Stage Temperature Time Initial Denaturation 95°C 12 min Denaturation 95°C 30 sec Annealing 64°C 30 sec Extension 72°C 30 sec Number of cycles 40 Final Extension 72°C 10 min 4°C ∞ Following amplification, the samples are centrifuged briefly and put on ice.