TITLE: n-PCR for a single sample
AUTHORS: Matias Ruggieri, Carolina Berini, Nicolas Ducasa, Miroslav Malkovsky, Paul Fisch, Mirna Biglione

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">HTLV-1 diagnosis using PCR is based on the amplification of the viral DNA sequences. This is usually conducted using a nested PCR which involves two rounds of DNA amplification reactions targeting the viral polymerase gene. In this case, we also add in the second round primers to amplify the human actin gene.</div></div>

[STEPS]
?. Equipment and MaterialsPipettes10µl presterilized filter tips20µl presterilized filter tips200µl presterilized filter tips1000µl presterilized filter tipsThermal Cycle (we used TProfessional TRIO)Electrophoresis equipment
?. Reagents and ChemicalsTaq DNA Polymerase kit (1000 U), by Qiagen, Catalog #: Cat No./ID: 201205Buffers (PCR Buffer and CoralLoad PCR Buffer)MgCl2Q solutionTaq polymerasedNTPsNuclease free H2OForward (F) and reverse (R) primersAgaroseMarkers: 50pb and/or 100pb (Thermo Fisher Scientific, Germany)
?. PCR summary protocol Primary PCR/ Nest-1 PCR should be done using PolEF and PolER  primers.For each group of samples include positive control (DNA from MT2 cell line) and a negative control of PCR mix without a DNA template.Nest-2 PCR should be done using the primers PolIF and PolIR. Also the primers Actin F and Actin R are necessary to amplify human Actin gene.Once the PCR run is complete, remove tubes and briefly centrifuge to spin down products.Store PCR tubes in the fridge or freezerRun the samples on 2% Agarose gel electrophoresis using gel red nucleic acid stain.Score results as presence or absence of bands comparing with control bands.
?. PCR ProtocolFor each run, at least two controls (1 positive and 1 negative) should be included in addition to the samples. Prepare the PCR master mix calculating enough volume for number of the samples +8%, to account for pipetting loss and dead volume. E.g. for 22 samples, one positive control and one negative control (total of 24 tubes), make a master mix for 26 reactions.Master Mix for Nest-1 PCR (10X PCR buffer, dNTPs, MgCl2, primers, Taq polymerase and water)Master Mix for Nest-2 PCR (10X CoralLoad PCR Buffer, dNTPs, MgCl2, primers, Taq polymerase and water)
?. eparation of PCR (Nest-1 PCR)  ABCD1Initial ConcentrationFinal ConcentrationFor 1 sample2PCR Buffer10x1 X2,5 ul3Q solution5x1X5 ul4Mgl225mM2,5mM2,5 ul5dNTPs2mM0,2mM2,5 ul6PolEF10mM0,3mM0,75 ul7PolER10mM0,3mM0,75 ul8Taq polymerase0,5 units0,1 ul9DNA sample5 ul (20 ng/µl)10Wateradd to a final concentration of 25 ulPreparation of PCR (Nest-2 PCR)  ABCD1Initial ConcentrationFinal ConcentrationFor 1 sample2CoralLoad PCR Buffer10x1 X2,5 ul3Q solution5x1X5 ul4Mgl225mM2,5mM2,5 ul5dNTPs2mM0,2mM2,5 ul6PolIF10mM0,3mM0,75 ul7PolIR10mM0,3mM0,75 ul8Actin F10mM0,3mM0,75 ul9Actin R10mM0,3mM0,75 ul10Taq polymerase0,5 units0,1 ul11First reaction product2 ul12Wateradd to a final concentration of 25 ul
ABCD1Initial ConcentrationFinal ConcentrationFor 1 sample2PCR Buffer10x1 X2,5 ul3Q solution5x1X5 ul4Mgl225mM2,5mM2,5 ul5dNTPs2mM0,2mM2,5 ul6PolEF10mM0,3mM0,75 ul7PolER10mM0,3mM0,75 ul8Taq polymerase0,5 units0,1 ul9DNA sample5 ul (20 ng/µl)10Wateradd to a final concentration of 25 ul
ABCD1Initial ConcentrationFinal ConcentrationFor 1 sample2CoralLoad PCR Buffer10x1 X2,5 ul3Q solution5x1X5 ul4Mgl225mM2,5mM2,5 ul5dNTPs2mM0,2mM2,5 ul6PolIF10mM0,3mM0,75 ul7PolIR10mM0,3mM0,75 ul8Actin F10mM0,3mM0,75 ul9Actin R10mM0,3mM0,75 ul10Taq polymerase0,5 units0,1 ul11First reaction product2 ul12Wateradd to a final concentration of 25 ul
?. Primers  ABC1RoundName5'-3' oligonucleotide sequence2Nest-1 PCRPolEFTTTAGGTGCCCAAACTGGAG3Nest-1 PCRPolERGCAGGATATTGGAAGCCTCAG34Nest-2 PCRPolIFGCCCTCATGCCAGTGTTTAC5Nest-2 PCRPolIRCCTGGAGATGGGATCAGGTAG6Nest-2 PCRActin FATCGAGCACGGCATCGTCACCAAC7Nest-2 PCRActin RGTTGAAGGTCTCAAACATGATCTG
ABC1RoundName5'-3' oligonucleotide sequence2Nest-1 PCRPolEFTTTAGGTGCCCAAACTGGAG3Nest-1 PCRPolERGCAGGATATTGGAAGCCTCAG34Nest-2 PCRPolIFGCCCTCATGCCAGTGTTTAC5Nest-2 PCRPolIRCCTGGAGATGGGATCAGGTAG6Nest-2 PCRActin FATCGAGCACGGCATCGTCACCAAC7Nest-2 PCRActin RGTTGAAGGTCTCAAACATGATCTG
?. PCR Cycling conditionsNest-1:Pol EF + Pol ER  ABC1Temperature (°C)Time2943 min39410 sec40 cycles45420 sec57045 sec6705 min74Hold        Nest-2:Pol IF + Pol IR + Act F + Acr R            ABC1Temperature (°C)Time2943 min39410 sec40 cycles45220 sec57045 sec6705 min74Hold
ABC1Temperature (°C)Time2943 min39410 sec40 cycles45420 sec57045 sec6705 min74Hold
ABC1Temperature (°C)Time2943 min39410 sec40 cycles45220 sec57045 sec6705 min74Hold
?. Preparation and storage of reagentsStock reagents for PCR (10X buffer, dNTPs, MgCl2, Q solution and primers) and enzymes should be stored at -20°C.Stock reagents should be diluted into working concentrations and aliquoted at -20°C into separate tubes.Working aliquots of sample and control DNA as well as primary PCR (Nest-1) products must be kept at -20°C for long-term storage but can be kept in the fridge for daily use.