Murtaugh Laboratory Procedures 2004 Real-Time PCR Protocol 7500 SYBR Green Assay Colleen Finnegan April 2006 Materials: 2X SYBR Green Master mix #4309155 Forward Primer 5 µM Stock Reverse Primer 5 µM Stock Template cDNA Sample Rnase/Dnase Free Water Mix together on ice (Reagent volumes are for one 15uL reaction): 7.5 µl of 2X Mastermix 0.3 µl Forward Primer 0.3 µl Reverse Primer 6.3 µl Add DNase/RNase free water For a total volume of 14.4 µl Place 14.4 µl of combined reagent mix in an optical plate or tube and add 0.6 µl (6-10ng) template cDNA. SYBR Green Real Time PCR with the ABI 7500 Machine 1. Log into the 7500 Machine Murtaugh Style. What’s another word for pirate treasure? 2. After pipetting mastermix/sample into 96-well plate/tubes (including No Template Control and Positive Control samples) seal with an optical tape or flat cap. 3. Turn on and load the plate into the 7500 machine. 4. Open ABI SDS Software and Select a New File. 5. Check that the following are selected: Absolute quantification, 96-well Plate, and ‘FinneganSYBR’ template from the drop down menu. Click Finish. Machine should say Connected to Template in the lower right corner of the file. Absolute Quantification and Relative Quantification Files are NOT Interchangable 6. Label wells according to plate set up. Do not omit wells in this step, omit after the run is complete with the Well Inspector. 7. Using well inspector, label the sample names if desired. 8. Next, go to the Instrument tab. Check the thermocycler conditions: 10 min. at 95 50 Cycles 15 sec. at 95 1 min. at 60. Add Dissociation Step Other Instrument checks must include: √ Reaction volume (15ul) and √ 9600 Emulation Mode 9. Before starting the run, save your file (as .sds) TO THE DESKTOP. The machine will not run a template file. Murtaugh Laboratory Procedures 2004 10. Click Start. 11. When the Run is complete a window will appear …Successful. 12. Re-Save file 13. To Analyze the Plate. Save file to Flashdrive and transfer to PC with SDS Software. Open File. Click upper left corner to highlight all the wells.
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