To evaluate the efficiency of the different types and different amounts of blocking primers, artificial rDNA mixtures were created. First PCR amplification were performed on krill and Pyramimonas DNA with the universal 28S rDNA primers Short28SF and Short28SR (Table 1 ) as 25 μL reactions with 0.25 μL of each oligo (10 μM), 0.25 μL dNTP, 2.5 μL 10×, 0.1 μL Platinum Taq DNA Polymerase High Fidelity (Invitrogen) 5 U. μL-1, 1 μL Mg2+ and 5 μL DNA (~10 ng. μL-1).
The PCR products were then cloned using the TOPO TA Cloning system competent cells (Invitrogen). Transformants were screened using blue/white selection on LB-agar containing X-Gal and 10 mg.mL-1 ampicillin or kanamycin. White or light blue colonies were picked for plasmid isolation, reincubated overnight in LB medium containing antibiotics and plasmids were then extracted using the Ultraclean miniplasmid extraction kit (Mo Bio, Carlsbad, CA, USA). Plasmids were linearized using HindIII restriction enzyme and the yield quantified using Picofluor 8000-004 (Turner Designs) and samples stained with Quant-iT PicoGreen ds DNA reagent (Molecular Probes).
Samples were thus mixed to contain a 100-fold and a 1000-fold excess of target rDNAs (krill 28SrDNA) compared with nontarget rDNAs (Pyramimonas sp.). A sample containing only krill DNA was used as a control.
Amplification with blocking primers were performed as 25 μL reactions with 0.25 μL of each of the universal 28S rDNA primers Short28SF(10 μM) and Short28SR(10 μM), 0.25 μL dNTP, 2.5 μL 10×, 0.1 μL Platinum Taq DNA Polymerase High Fidelity (Invitrogen) 5 U. μL-1, 1 μL Mg2+ and a variable amount of blocking primer and rDNA. PCR thermal cycling conditions were: 2 min at 94°C; 40 cycles of 10 s at 94°C, 30 s at 59°C, 30 s at 68°C; and finally 5 min at 72°C.
Blocking efficiency was assessed by fragment analysis of the fluorescently labelled PCR products. Fragment analysis separates a mixture of DNA fragments according to their sizes and is much more sensitive than standard gel electrophoresis. The analysis was performed on an Applied Biosystems 3130 xl Capillary Electrophoresis (CE) Genetic Analyser and results were analyzed with Peak Scanner Software 1.0 (Applied Biosystems).
The PCR products were then cloned using the TOPO TA Cloning system competent cells (Invitrogen). Transformants were screened using blue/white selection on LB-agar containing X-Gal and 10 mg.mL-1 ampicillin or kanamycin. White or light blue colonies were picked for plasmid isolation, reincubated overnight in LB medium containing antibiotics and plasmids were then extracted using the Ultraclean miniplasmid extraction kit (Mo Bio, Carlsbad, CA, USA). Plasmids were linearized using HindIII restriction enzyme and the yield quantified using Picofluor 8000-004 (Turner Designs) and samples stained with Quant-iT PicoGreen ds DNA reagent (Molecular Probes).
Samples were thus mixed to contain a 100-fold and a 1000-fold excess of target rDNAs (krill 28SrDNA) compared with nontarget rDNAs (Pyramimonas sp.). A sample containing only krill DNA was used as a control.
Amplification with blocking primers were performed as 25 μL reactions with 0.25 μL of each of the universal 28S rDNA primers Short28SF(10 μM) and Short28SR(10 μM), 0.25 μL dNTP, 2.5 μL 10×, 0.1 μL Platinum Taq DNA Polymerase High Fidelity (Invitrogen) 5 U. μL-1, 1 μL Mg2+ and a variable amount of blocking primer and rDNA. PCR thermal cycling conditions were: 2 min at 94°C; 40 cycles of 10 s at 94°C, 30 s at 59°C, 30 s at 68°C; and finally 5 min at 72°C.
Blocking efficiency was assessed by fragment analysis of the fluorescently labelled PCR products. Fragment analysis separates a mixture of DNA fragments according to their sizes and is much more sensitive than standard gel electrophoresis. The analysis was performed on an Applied Biosystems 3130 xl Capillary Electrophoresis (CE) Genetic Analyser and results were analyzed with Peak Scanner Software 1.0 (Applied Biosystems).
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