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troubleshoot pcr 6-29-13
2014-08-29
azim58 - troubleshoot pcr 6-29-13 6-29-13 I've been having some trouble reproducing results of my pcrs. Here are some factors that might affect the quality of a pcr. -how many times the template has been frozen and thawed --suspected best condition: frozen and thawed fewest times -whether or not the template has denatured before adding the polymerase --suspected best condition: denatured 2 min before adding polymerase -how long the sample is on the cold block before applying thermocycling conditions --suspected best condition: sample on cold block for minimum amount of time -whether or not the pcr tubes are vortexed and spun down before applying thermocycling conditions --suspected best condition: pcr tubes vortexed and spun down before thermocycling I'd like to write out my protocol and follow it by checking off each item as it is completed. I will test the 4 different conditions described above with 5 different groups. Group 1 Old template -Add the following reagents to pcr tubes in the following order --40.5 uL H20 --5 uL 10X buffer --1 uL 100 mM dNTP --1 uL SMC forward primer --1 uL SMC reverse primer --0.5 uL template (1: old cDNA, 2: old cDNA, 3: old cDNA, 4: H20) -Vortex the pcr tubes and spin them down -start "hs dyn tdwn" thermocycler protocol -Add samples to thermocycler after a high temp has been reached -Allow the samples to denature for 2 m -Add the dynazyme polymerase and allow the thermocycler protocol to continue. Group 2 No denaturation -Add the following reagents to pcr tubes in the following order --40.5 uL H20 --5 uL 10X buffer --1 uL 100 mM dNTP --1 uL SMC forward primer --1 uL SMC reverse primer --0.5 uL template (1: new cDNA, 2: new cDNA, 3: new cDNA, 4: H20) -Vortex the pcr tubes and spin them down -start "hs dyn tdwn" thermocycler protocol -Add samples to thermocycler after a high temp has been reached -Do not allow the samples to denature for 2 m and the dynazyme polymerase. Then allow the thermocycler protocol to continue. Group 3 Long cold block -Add the following reagents to pcr tubes in the following order --40.5 uL H20 --5 uL 10X buffer --1 uL 100 mM dNTP --1 uL SMC forward primer --1 uL SMC reverse primer --0.5 uL template (1: new cDNA, 2: new cDNA, 3: new cDNA, 4: H20) -Allow the sample to sit on the cold block for 30 min -Vortex the pcr tubes and spin them down -start "hs dyn tdwn" thermocycler protocol -Add samples to thermocycler after a high temp has been reached -Allow the samples to denature for 2 m -Add the dynazyme polymerase and allow the thermocycler protocol to continue. Group 4 No vortex -Add the following reagents to pcr tubes in the following order --40.5 uL H20 --5 uL 10X buffer --1 uL 100 mM dNTP --1 uL SMC forward primer --1 uL SMC reverse primer --0.5 uL template (1: new cDNA, 2: new cDNA, 3: new cDNA, 4: H20) -Do not vortex the pcr tubes and spin them down -start "hs dyn tdwn" thermocycler protocol -Add samples to thermocycler after a high temp has been reached -Allow the samples to denature for 2 m -Add the dynazyme polymerase and allow the thermocycler protocol to continue. Group 5 All optimal -Add the following reagents to pcr tubes in the following order --40.5 uL H20 --5 uL 10X buffer --1 uL 100 mM dNTP --1 uL SMC forward primer --1 uL SMC reverse primer --0.5 uL template (1: new cDNA, 2: new cDNA, 3: new cDNA, 4: H20) -Vortex the pcr tubes and spin them down -start "hs dyn tdwn" thermocycler protocol -Add samples to thermocycler after a high temp has been reached -Allow the samples to denature for 2 m -Add the dynazyme polymerase and allow the thermocycler protocol to continue.
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