The QPCR Machine
You may also be interested in Quantitative PCR on the Roche LightCycler 480 at the Buck
Setting up your QPCR:
You'll need: QPCR plate(s), plastic adhesive cover for QPCR plate, filter pipette tips (200uL and 20uL ), multichannel pipette (5.5uL), singed up time on qPCR machine and any ingredients listed below:
1. Get a QPCR sheet for yourself (probably from just about anyone in the lab) and a QPCR plate to help you visualize the process.
2. Decide on your setup. For instance, if you have only one strain to test, you need eight(8) samples for that strain using its two combined primers, so you'll need eight rows of the QPCR plate and some number of columns (maybe 3) to replicate the data, see the picture below. In this case, YIR was our test strain, and cdc28 was our control strain. Four columns of eight rows of each strain were run with two columns of five rows for each strain as standards run as well. These will be explained below. It may also be helpful to highlight on your plate(s) which wells are which, ie red=YIR042c and black=CONTROL or something.
3. Now that you have the vision in your head, go grab an ice bucket and fill it with ice. Then go to the -20C freezer in the hallway and grab a tube of SYBRgreen or two from the top box in the rack (probably should ask to be shown which rack the first time). Then go to the -80C freezer and take out the standards and samples. These are in another box in another rack on the right half of the freezer, again, you should probably be shown where since labels never seem to make sense anyway.
4. THEN go to the
-20C freezer in the lab and get your primers. These primers are specially ordered QPCR primers , so if you don't have them, put everything back and go order them . The 10uM primer concoction needed for the QPCR (it makes a bunch, so you don't have to do it every time) is 400uL dH2O plus 50uL of each of the primers you ordered (and subsequently diluted to 100uM). It's possible you won't have your own stock of control primers, in which case you should order them and make it, but for now it's guaranteed that someone else in the lab has some, so ask (cdc28?).
5. Now that you have all these things on ice (tip: keep them organized!!!) you can make your master mixes. Now would also be a good time to go get the multi-channel pipette from the other room (Chris B's bench), you'll need one that can dispense 5.5uL.
6. Your master mixes should be 380uL of SYBRGreen and 38uL of your primer concoction. You'll have two probably (control and test). In general use the least that you will need which may be less than listed here; SYBRGreen isn't free.
7. Pour your first master mix into a multichannel well (found in a drawer on the right side of the chemistry bench on your right as you enter J337). Pipette 5.5uL into the appropriate wells of the QPCR plate. Repeat this process for all your wells, relative to where each primer belongs.
8. Now you need to load each well with its sample of 4.5uL each (five control standards, five test standards, 8 control samples and 8 test samples- your numer of samples and number of test strains per plate may vary). You'll have to do this one-by-one, so find a way to keep track of where you are AND USE A FRESH TIP EVERY TIME!!!
9.Once your plate is loaded you need to put the plastic cover on it. For now, use a sharpie or something to flatten it nicely on the top of the wells. Then proceed to the qPCR machine. It is recommended you take someone knowledgable with you for your first time.
NOTE: All of the above and below: Every use of "SYBRGreen" can be replaced by "Ssofast EvaGreen". The set up is exactly the same. SEE NOTE BELOW TOO!
QPCR MACHINE:
1. Use the provided gold-colored thingy to flatten the plastic cover onto your plate EXTREMELY WELL. Like seriously, smash that down. Then place the plate into the qPCR machine in the appropriate orientation (not upside down and flipped around...)
2. Start the iCycler software, click YES then OK on the irritating error message. The program will open into the library tab, subsection "protocol". Go to Kristen's folder and select "standard" as the protocol, then go to the plate setup subsection and select your plate setup. NOTE: For Ssofast EvaGreen, use the protocol called "vmsso.tmo", and keep your same plate set up. This will vary based on how you set up your plate; most likely, though, someone has a set up just how you need it, so pick your co-worker based on this possibility and have them explain it and make sure it's right. Then, when everything is confirmed correct, tell it to begin the run. It will prompt you to enter the well volume - make sure to turn this down since you've only added 10 uL per well and the default is 50. Heads up though, the machine hates you if you enter 10, so tell it 15.
3. Tell it to start, go back to your bench and play with tanagrams until it said it'd be done, then go back to the machine and pop it open, remove your plate and stick in your flash drive.
4. On the page with a table of data and graph, you have to take your data off yourself, so click the top-left box in the spreadsheet and do a ctrl+c, then open excel and ctrl+v and save your sheet to your flash drive. Also helpful are the reports, which give you the spreadhseet in a Word format AS WELL AS the graph, depending on which report you pick. You may want one of those too.
5. Make sure to EJECT YOUR FLASH DRIVE so the ancient computer doesn't fry your data and that starcraft.iso you had on there...
6. Now you're done! BUT MAKE SURE TO TURN OFF THE LAMP AND MACHINE IF YOU ARE DONE!!! The lamp switch is on top in the back of the machine and the machine switch is on the side. Don't turn off the computer as per the instructions printed on the computer.
SYBR Green: P/N: (Roche, 04 887 352 001, -20C freezer)