QPCR

Real Time qPCR

Materials

• cDNA
• SyberGreen PCR Master Mix Applied Biosystems
• 384 well qPCR plate
• Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). Generate primers according to Primer Design for qPCR

Plate Preparation

1. Book 2h on qPCR machine by going to http://slimprimplus.uthsc.edu/SlimPrim/index.php?action=workflow&module_id=4&sub=lc480logbookrequest
2. Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
3. Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
4. Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA;s
5. Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
6. Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 10 uL SYBR green + 5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 100 uL SYBR Green + 50 uL Primers.
7. Using the repeater multichannel pipettor put on 2 or 3 tips (depending on your plate arrangement) and set to suck up 122 uL and dispense 15 uL per well.
8. Dispense 15 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
9. Using the snap clip multichannel add 5 uL of cDNA to each applicable well. The width between tips should be 2 wells, so you should be able to add to columns 1/3/5/7... in one group and rows 2/4/6/8... in another group. If you add the cDNA to the top of the well and the primer mixture to the bottom you dont need to change tips.
10. Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.
11. You can prepare the plate ~3h beforehand, keeping it at 4C
12. Immediately before the run spin the plate briefly in the swinging bucket centrifuge.

Run Protocol

1. Open LightCycler software.
2. Click new experiment.
3. Choose SYBR Green option from drop down and make sure that the correct block is in place (it will say 96 or 384).
4. Click apply template.
5. Go to templates>run templates> bridges SYBR Green 384.
6. Load plate and click 'start run'.
7. This protocol is set to do the following:
8. Activate- 1 cycle @95 degrees for 10s.
9. Amplify- 45 cycles @95, 60 and 73 degrees for 15s, 15s and 10s, respectively.
10. Melt- 1 cycle @95, 40, 65, 95 and 40 degrees for 1m, 1m, 1s, continuous and 10s, respectively.

Calculations

see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations