troubleshoot pcr 6-29-13
2014-08-29azim58 - 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
- -suspected best condition: sample on cold block for minimum amount of
- whether or not the pcr tubes are vortexed and spun down before applying
- -suspected best condition: pcr tubes vortexed and spun down before
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
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
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
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
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