Cre genotyping protocol

Primers:

ColIICre F            TCG ATG CAA CGA GTG ATG AGG

ColIICre R            GAA ACC ATT TCC GGT TAT TCA ACT TGC

 

Col10 F                 CTT CCT GTC AAG CTC ATC C

Col10 R                TAG GAT TGC TGA GTG CTC C

 

Protocol:

prepare genomic DNA by phenol/chloroform extraction and determine concentration

.              |

dilute primers to 10 pmol/μl using ultrapure dH2O

.              |

set up a master mix (on ice) for each primer (per reaction) as follows:

1μl forward primer

1μl reverse primer

13μl H2O

25μl SYBR Green Master Mix

.              |

add template DNA to MicroAmp wells. Add 50ng DNA in 10μl H2O

.              |

add the primer master mix to each well (40μl per well) of a white 96-well plate (BioRad)

.              |

seal plate with BioRad caps or MicroAmp Optical Adhesive covers

.              |

run PCR program:

50°C                      2:00

95°C                      10:00

95°C                      00:15  ——|

60°C                      00:30         |           x 30                                                                                                 

72°C                      01:00  ——|

 

Note

Use filter tips for all pipetting steps.

 

Data analysis

Use the melting curves to assess the PCR run. A single distinct peak should be seen, indicating a single PCR product. Water blanks produce noise of many peaks. More than one peak in the trace indicates potential contamination.

Calculations: set a cycle threshold manually at the qPCR machine to where the curve starts rising on the plot. Use the Ct values exported for each sample to calculate:

dCt = Ct(Cre) – Ct(colX)

 

Calculate ddCt using the wild type control as the calibrator:

ddCt = dCt(samples) – dCt(calibrator)

 

To find the linear fold change between the samples and the control, calculate:

2(-ddCt)