Allyson Luber

September 17th, 2019

I. Electrophoresis of PCR products

1. We took out our PCR tubes and let them thaw
2. After thawing, one of our group members dotted out 13 loading dye dots (~1μl) on a sheet of parafilm (our group did 13 dots compared to the normal 16 dots because there’s only 3 of us)
3. Next we pipettted 3 μl of each PCR product into its own dot, using a 10 μl pipette
4. Loaded all dots into the gel (set pipette to 5 μl)
5. Ran the gel at 130 volts for 30 minutes

Order of lanes on Gel Electrophoresis plate:

(Top)

1. Negative control
2. RG- 01
3. RG-02
4. RG- 04
5. EVR-01
6. EVR-02
7. EVR-03
8. EVR-04
9. AEL-01
10. AEL-02
11. AEL-03
12. AEL-04

15. Ladder

II. Clean-Up of PCR products for sequencing- ExoSAP

1. Labeled new 0.2 μl PCR tubes with each of my sample codes (label twice on the side of tube)
2. Make the ExoSAP Master mix – one per table

• The volume of the Mater mix was calculated based off how many PCR clean-ups our table did
• Put reagents on ice
• Pipetted 7.5 μl of each PCR product into a clean, labeled 0.2 μl PCR tube
• Made the the ExoSAP master mix, keeping the reagents on ice while it was made
• Pipetted 12.5 μl of master mix into each PCR product tube
• Placed the tubes in thermocycler and started the ExoSAP program
• After program was complete, the PCR tubes were placed in a labeled tube rack and into the freezer

Master Mix volumes:

Master mix:                                         Rxn: 1                           Rxns: 13

1. H2O                                           10.59 μl                            138 μl
2. 10x buffer (SAP 10x)                 1.25 μl                              16.3 μl
3. SAP                                            0.44 μl                              5.7 μl
4. Exo                                             0.22 μl                              2.9 μl

Master mix total:                                 12.5 μl                            162.9 μl

PCR Product

PCR                                                    7.5 μl

Total Cleaned-up volume                    20.0 μl

End result of fish PCR 

(Last 4 lanes to the right, before the ladder, are mine)