MY MICROSATELLITE DILUTION AND PLATE PROTOCOLS FOR 3730
PCR PLATES
Original PCR out of PCR machine (If I am going to run the samples in the 3730 that day, I put the PCRs in the fridge until I am ready. If I am going to run them later than that, I put them in the freezer)
transfer 3µl of DNA from each PCR into a PCR MIXTURE plate (I always run 5 microsatellites per lane - less is a waste of money and if I run 6, I always have one microsatellite that fails to show up)
Transfer
3µl of DNA from the PCR MIXTURE plate
GEL PLATE THAT GOES IN THE MACHINE
Transfer 8µl of mix to each well
FORMAMIDE – SIZE STANDARD MIX
- 880µl of HiDi Formamide (from ABI - keep in freezer)
-16.5µl of Tamra 500 Size Standard (from ABI - keep in fridge)
- keep plate on ice until you denature the DNA
- put grey rubber septae into the wells of the plate
- denature for 2 minutes on a hot block or in a PCR machine at 96˚C
- snap cool on ice until the plate is cool
- put the plate into the plate holder apparatus and place in the stacking dock of the machine
- the status can be found in the bottom right corner of the window on the computer screen
- If the machine is not already running, press the green arrow button in the top left of the computer screen
- transfer your sample sheet to the computer via USB drive
- a screen with a lot of options down the left side should
be visible on the computer. If it
isn’t, bring it to the front (it may be hiding at the bottom of the screen)
- click on plate manager to import the sample sheet
-
click import and navigate to the USB drive to find your file
- click on run scheduler
- click
Search A….
- Click
Find All
- Scroll
down to find the sample sheet
- Click
Add
- Click
Done
SAMPLE SHEET
- the sample sheet must be saved as a .txt file
- no
symbols or spaces (only letters and numbers) can be used in the sample name
