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.