Pooling Protocol

~Use this pooling protocol after TS PCR and check gel verification in preparation for index PCR~

Note: you can only pool TS-PCR product together if you have used a unique primer in the first round of PCR (e.g., markers for a first round PCR of the wingless gene and elongation factor gene are completely different and can be separated bioinformatically)

We are pooling TS PCR products by in-line barcode (ie: ARF1 and MCO).

3 gDNA plates → 2 TS PCR plates [ARF1 A, B, or C]

    [MCO A, B, or C]

3 gDNA → 6 TS PCR plates → 1 pooling group → 2 index PCR plates

  1. Determine pooling groups:
    • Plates are grouped based on criteria:
      • Each group ideally has A, B, and C. The group can also consist only of one or two of these in line barcodes/plates but there cannot be duplicates. 
      • By sampling method (eg: beating, canopy, leaf litter, etc)
  2. Create a plate map for the pooled plate:
    • Add a new sheet and label it appropriately with the group you are working on
    • Label page with your name and date
    • Make sure to note which plates you are using 
    • Insert screenshots of the plate maps and gel images to the sheet
    • You can copy and paste the template plate map to your new sheet/tab and fill in your information appropriatly
    • Make sure you have checked the redo plate map and gel image for any failed bands 
    • If there are any fully empty wells, designate one to pool the negative controls. If there is not an empty well in this plate, find an empty well in another pooled plate and make sure to label it and write it down so we all know what’s up. 
    • Last check: if there are any wells with only one sample and that sample has a sale band (ie: adding 2 uL), change volume to 4ul to meet be minimum volume needed in each well. 
  3. Fill in plate map: use plate maps and gel images to determine empty wells, negative control (nc), and volume of sample to combine based on band strength
    • plates are pooled together based on location/well and the volume is determined by the gel band strength, which indicates DNA concentration. The varied volumes of sample used is to yield relatively even concentration of each sample in the combined well. 
      • Strong/dark band : 2 uL
      • Medium band : 4 uL
      • Weak/light band : 6 u
      • ** there must be at lest 4uL TS PCR product in each pooled well**
      • Note: This pools samples based on relative band strength, so that the concentration of DNA from each well is proportional. In other words, if you want to pool equal molar amounts of TS-PCR product together, you take 2 ul of a dark strong band and put it with 4 ul of a medium band and 6 ul of a weak band in a new plate.
  4. Print plate maps. One to use and mark up as you pool. One that’s nice and clean to be taped into the lab notebook. 

Now you are ready to put on gloves and pool!

  1. Prepare and clean workspace: 
    • put on gloves
    • Wipe down bench with 10% bleach
    • Locate TS PCR plates needed for pooling (should be grouped in freezer with colored tape). Remember you probably need the mixed redo plate along with the three original plates. Set plates in plate racks and let thaw on the bench top
  2. Label a new PCR plate with:
    • Your name/initials
    • Date
    • Plate group name
    • “pooled”
    • In-line barcode/marker: ARF1 A, B, C or MCO A, B, C
  3. Gather your printed plate map, sharpie/pen, filtered p10 tips, p10 and p2 micropipettes, p10 multichannel pipette
    • Set p2 to 2ul
    • Set one p10 to 4 uL
    • Set another p10 to 6 uL 
    • Note: this is not essential to the process but it saves time. Also, it’s helpful to label these with different colored tape
  4. Vortex and spin down TS PCR plates
  5. Now its time to focus and pipette:
    • Do whatever you need to set yourself up for success. 
    • You can go row by row, plate by plate, or any other method that works for you. 
    • Ex: mark off each well after you’ve added it to the pooled plate
    • Double check you’ve added any redo samples as needed and put the negative control in its appropriate place. 
    • Reseal all plates with adhesive aluminum foil top. 
    • Return all plates in the freezer. Pooled plate should go in the rainbow tape bag, TS PCR plates should go w the other plates we are  “done” with, mixed redo plate should go back with the “to do” plates. 
    • Clean up: Wipe down bench again and put things away.
  6. Clean up: wipe down bench again and put things away
  7. Now you are ready to run Index PCR protocol