Genotyping

PCR Protocol – Long Version

1. Get ice.  Everything you use (except DNA samples) needs to be kept on ice the entire time.

2. Access the Google sheet:

   1. Check Drive or us the link: https://tinyurl.com/pcrsheet

3. Determine which gene to genotype

   1. Your group has been assigned a specific gene. Check the Google sheet to determine which gene you are responsible for.
   2. Goal is to have 0% remaining
   3. Navigate to that sheet (or click the hyperlinked gene name)

4. Select and Organize Tail Samples

   1. Ear tag is the mouse number – keep track of which mouse the tail snip is from
   2. Scroll down until you reach the section of ungenotyped mice (empty boxes in the gene column) 
   3. Choose at least 10 ungenotyped tails and write down the ear tag # in your lab notebook. (you can successfully do up to 60).
   4. Choose at least 1 control – it’s best practice to pick at least 2 (one WT and one with gene of interest).  Write down the ear tag # in your lab notebook. 
   5. Locate the actual DNA tail samples in the radioactive fridge.  These are kept in Eppendorf tubes in boxes with the gene of interest at the label.
   6. Organize the samples into groups of 8.  Write down the groups in your lab notebook.  At all times, keep track of your mouse numbers because if you get confused, the data is useless.
   7. Label PCR tubes (snap strip) with mouse numbers of each group of 8.  
   8. Vortex/spin down the DNA samples.  Set aside.

5. Navigate to the “Protocols” tab on the Google Sheet

   1. Scroll until you find the recipe for the gene of interest
   2. Go to the -20’ freezer and grab the associated reagents bag for the gene of interest.  Allow all reagents to thaw. Keep the polymerase/Taq in the ice.  This must remain as cold as possible throughout.
   3. Determine in the number of reactions (10% more than the number of samples you have.  E.g. 16 samples should have 18 reactions, so you have a little wiggle room). 
   4. Input the number into the sheet in the YELLOW BOX. (The values underneath should now automatically update).

6. Create Master Mix

   1. Label and empty Eppendorf tube “Master Mix”

   2. Add the PURPLE BOX reagent amounts in the order they appear (biggest to smallest volume-wise)

      1. Vortex and spin each reagent before adding
      2. Pipette mix each time a new reagent is added
      3. Keep the master mix on ice while moving onto the next reagent (Key is to keep it cold).
      4. Taq or Polermase should not be added until the very end.
      5. Vortex and spin the Master Mix after everything is added.

7. Add Master Mix to Individual PCR tubes.

   1. Each tube receives the volume in the GREEN BOX. Keep these on ice!

8. Add DNA sample to each PCR tube.

   1. Keep track of which sample goes in each tube. 
   2. Add the volume in the BLUE BOX.
9. Vortex and Spin. The reaction is ready to start.

10. Run in PCR Machine.

    1. Put in the PCR machine.  Make sure all the tubes fit in the associated holders or else some won’t work)
    2. Double check the lids of the PCR tubes are on TIGHT or else it will all evaporate away.

    3. Once loaded, select the protocol on the screen.

       1. Open Method
       2. Genotyping (Nr4a3 is separate)
       3. The method name is on the Google Spreadsheet in the Protocol tab.  (PCR machine protocol name).
       4. Let it run.
       5. Make sure to remove samples when it is done (they take anywhere from 45 minutes to 2 hours) so it doesn’t sit all day and waste energy.

11. Prepare gel (3% agarose)

    1. If running less than 20 samples:

       1. Get a 125 ml beaker
       2. Add 2.25 g of Quick Dissolve Agarose (dry shelf)
       3. Fill with 75 ml of 1x TAE buffer (near the electrophoresis machine)

    2. If running more than 20 samples

       1. Get 250ml beaker
       2. Add 3 g of Quick Dissolve Agarose (dry shelf)
       3. Add 100 ml of 1x TAE buffer (near the electrophoresis machine)

    3. Microwave for 3 minutes at half power (set power level to 5)

       1. Make sure it doesn’t boil over
       2. Remove from microwave with hot pad – it is extremely hot

    4. Prepare mold

       1. If doing less than 20 samples, use the smaller mold; if more, use the bigger mold
       2. Make sure to place the comb so you have wells at the end (2 rows if needed – use the 30 well comb; the 40 well comb is too small)

    5. Add Nucleic Acid stain to the molten agarose

       1. 5ul per 100ml of gel
       2. Located in the radioactive freezer in the top shelf of the door.

    6. Pour into the mold and allow to cool for at least an hour

       1. Do not let sit out for too long (more than 3 hours) or else the gel will dry out

12. Prepare samples for loading

    1. Once the PCR machine is done running the protocol, remove the samples

    2. Add 1ul of loading buffer to each sample

       1. Loading buffer is in the media freezer, it is blueish/green.
       2. Pro-tip: there are two special pipettes near the electrophoresis machine.  These are the PCR pipette because the 10ul pipette uses the p200 tips and the 30ul pipette uses the p10 tips.  Use that 10ul pipette to add the loading buffer so it doesn’t clog. Otherwise, you will be switching tips every sample.
       3. Vortex and spin until evenly mixed.  Samples should be blue now.
    3. Remove the DNA ladder (same location as the loading buffer in the freezer) and allow to defrost

13. Run the gel

    1. Orient the electrophoresis machine. (98% of the time this is already done but incase it is wrong: Black in back.  There is a black end and red end.  The black end should be farthest away from you on the counter.  If you flip this, your sample will run straight off).
    2. Place your hardened gel with the wells farthest away from you (similar to the black side of the machine – your samples run from black to red so you want them to run through the gel instead of running straight off).  
    3. Fill the electrophoresis machine with 1x TAE buffer up to the fill line on the side of the machine.  Make sure there are no air bubbles in the wells.
    4. Once again, keep track of which samples you load into each well.

    5. Load the volume specified in the ORANGE BOX.

       1. Do them in groups of 8 and put a 4ul of DNA ladder between each set of samples.

       2. Pro-tips: 

          1. use the 30ul pipette that takes 10ul tips because there is less capillary action in those tips so you will have less blowouts.
          2. If you are ejecting sample and nothing is leaving the tip, stop pushing!  Release the pressure, lift the sample out of the water and try again. Most likely, you have a small air bubble at the entrance to the pipette tip that is holding your sample in. 
          3. Slowly pull your tip out as you load to make room for your sample.  The tip takes up over half the available volume so get it out of the way as you fill up the wells.
    6. Add Nucleic Acid stain to the 1x TAE buffer in the electrophoresis machine on the RED side (closest side to you).  This stain runs opposite to DNA so it will run through the gel the other direction and illuminate your samples.
    7. Place the lid onto the electrophoresis machine (careful not to disturb the liquid inside.)
    8. Turn on the electrophoresis machine and turn it to 120V.  It might start beeping loudly halfway through – turn the knob down until the machine is happy.
    9. Run for 20 minutes.  Turn off the machine.

14. Image the gel.

    1. Remove the gel from the electrophoresis machine and carefully transfer to the scanner.
    2. Open DiDigit on the computer.

    3. Change exposure to 2x and image.

       1. If it is too dark, change is 3x and keep increasing the exposure until you can clearly make out the bands of your sample).  Too light (overexposure) then reduce the number. 2x usually works but you will have to play around with this.
    4. Save the image to a safe location (you can follow the path that it got saved into in the settings of DiDigit and you should copy that file onto a flash drive and save it on a personal computer or online in Box.)

15. Genotype

    1. Google Sheet Protocol tab has band locations.  The DNA ladder is in 100bp increments with 1000bp and 500bp being the two brightest bands.
    2. Figure out the genotype of each sample and send your best guess / image to Jacob.  He will verify and add give you a prize if you got 100%.
    3. You are done.  Good job.

1. Troubleshooting (Didn’t work?  Yeah that happens.)

   1. Did your ladder show up:

      1. Yes: Proceed to b.
      2. No: You probably ran it backwards.

   2. Did your controls work?

      1. Yes: Samples are dirty. Proceed to c.
      2. No: Proceed to d.

   3. Nanodrop them to check concentration and the A260/280

      1. If you don’t know how to do this: ask a Nr4a group member
      2. Concentration should be between 30-100ug and the A260/280 should be around 1.8.  If these are off, then there is a DNA purification/concentrating kit.

   4. Did you do it on ice/keep polymerase cold throughout?

      1. Yes: proceed to e.
      2. No: You done and messed up A-A-Ron.  Polymerase might be bad. Talk to Jacob.

   5. Did you do the right protocol? Right reagents?  Right PCR machine protocol?

      1. Yes: reagents are probably bad. Proceed to f.
      2. No, then there is the issue.  Redo it fixing the mistake.

   6. Did you have to make/add any new reagents because you used up the last of the one?

      1. Yes: this is the issue and double check you have the right concentration/reagent.
      2. No: Proceed to g.

   7. Has it worked in the past?

      1. Yes.  Uh oh.  Maybe just do it again?  
      2. No. Uh oh. Well then, maybe all the reagents are bad / DNA is bad / its all wrong.  This is the worst when it stops working.