Gel Electrophoresis and Exo-Sap Clean Up

 

Protocol:

Gel Electrophoresis of gDNA

  1. Pipetted 2 microliters of loading dye per sample on parafilm (10 total)
  2. Added 3 microliters of PCR product to each loading dye dots
  3. Pipetted about 5 microliters into following wells
    1. RA01
    2. RA02
    3. RA03
    4. JPCA
    5. JPCB
    6. JPCC
    7. JPCD
    8. LADDER
    9. JW01
    10. JW02
    11. JW03
    12. LADDER
  4. Placed lid on pre-setup gel electrophoresis box with DNA running to red
  5. Set to 145 volts then ran for about 15 minutes

Imaged using gel doc EZ imager

Results: Not bad


 

Gel Electrophoresis of PCR Products

Making Gel:

  1. Small Gel- .5 g agarose, 50 ml 1x TAE Buffer, 0.5 microliters diluted Gel Red per 50 ml gel

However, we just reused the gel from earlier. It was melted in microwave for about 25 seconds. Then we added 1 microliter of Gel Red

2. Poured hot gel into gel mold, added combs and let cool


Gel Electrophoresis of PCR Amplified DNA

  1. Pipetted 2 microliters of loading dye per sample on parafilm (10 total)
  2. Added 3 microliters of PCR product to each loading dye dots
  3. Pipetted about 5 microliters into following wells
    1. RA01
    2. RA02
    3. RA03
    4. JPCA
    5. JPCB
    6. JPCC
    7. JPCD
    8. LADDER
    9. JW01
    10. JW02
    11. JW03
    12. LADDER
    13. NONE
    14. NEGATIVE
  4. Placed lid on pre-setup gel electrophoresis box with DNA running to red
  5. Set to 145 volts then ran for about 15 minutes

Imaged using gel doc EZ imager

Results:

Use RA02 RA03 JPCC JPCD JW01 JW02 JW03


ExoSap PCR Clean-Up Protocol

 

 

 

DNA Extraction from Animal Tissue

Protocol:

  1. Recorded sample info and labeled 1.5 ml screw cap tubes with the following:
    1. JPCA – Escolar
    2. JPCB – Yellowtail
    3. JPCC – Black Snapper
    4. JPCD – Albacore Tuna
  2. Snipped 2-10 mg samples of each fish species (weighed the first one and approximated the rest), wiped blade with ethanol between each change of species
  3. Added 100 microliters of Extraction Solution and 25 microliters of Tissue Preparation to labeled tubes (solutions from Sigma REDExtract-N-Amp Tissue PCR Kit)
  4. Added samples to each tube
  5. Incorrectly incubated tubes at 95 degrees Celsius for 10 mins
  6. Removed tubes from heating block, allowed them to cool for 5 minutes
  7. Added 100 microliters of Neutralizing Solution from same kit
  8. Vortexed for 10 seconds
  9. Put samples on ice
  10. Made 10x dilution of gDNA- 18 microliters diH2O + 2 microliters of gDNA
  11. PCR Reagents for each tube (14 Reaction overall Master Mix)
    1.  Note: Pipette was somewhat wonky
    2. PCR Quality Water – 6.4 µl
    3. REDExtract-N-Amp PCR rx mix – 10 µl
    4. Forward Primer – 11.2 µl
    5. Reverse Primer – 11.2µl
  12. added 2 µl Diluted Tissue Extract to each labeled PCR tube
  13. Put into thermocycler with following settings
    1. 94 C – 4 minutes (initial denaturation)
    2. 30 Cycles of
      1. 94 C for 30 sec (denaturing)
      2. 52 C for 40 sec (annealing)
      3. 72 C for 1 min (extension)
    3. 72 C hold for 10 min (final extension)
    4. 10 C hold indefinitely