Lab 4- Gel Electrophoresis/ PCR Clean-up

To begin the electrophoresis of PCR product:

  • First, we thawed our PCR tubes
  • We dotted 16 loading dye dots (~1 ul) on a sheet of parafilm
  • We pipetted 3 ul of each PCR product on its own dot
  • We then loaded all dots into the gel (setting the pipette to ~5ul)
  • We ran the gel at 130 volts for 30 min

My PCR products were placed in lanes 9-13, beginning with KRS1 and ending with the negative control, respectively.

Clean-up of PCR products for sequencing- ExoSAP

  • First, we labeled new 0.2 ul PCR tubes with each of the sample codes
  • We then made the ExoSAP Master Mix
  • Lastly, we placed the PCR tubes into a thermocycler

ExoSAP PCR Clean-Up Protocol 

Recipe to clean-up one PCR reaction of 7.5 uL

Master Mix:                                  Rxn: 1          Rxns: 18

H2O                                                10.59 uL            190.62 uL

10x buffer (Sap 10x)                      1.25 uL             22.5 uL

SAP                                                0.44 uL               7.92 uL

Exo                                                0.22 uL               3.96 uL

Master Mix Total                    12.5 uL              225 uL

PCR Product:

PCR                                                7.5 ul

Total Cleaned-up Volume   20.0 uL


  • We determined the number of PCR clean-ups
  • We calculated the volume of Master mix needed
  • We put reagents on ice
  • We pipetted 7.5 uL of each PCR product into a clean, labeled 0.2 uL PCR tube
  • Then we made the ExoSap master mix, keeping the reagents on ice while it was made
  • We pipetted 12.5 uL into each PCR product tube
  • We placed the tubes in a thermocycler and start the EXOSAP program
  • After the program completed (~ 45 minutes), we placed the PCR tubes in a labeled tube rack and placed them in the freezer

    DNA barcode for our group, Queen Salmon.

PCR results after 16 hrs 5 min.


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