Rationale:

To find out to which cluster Ferranti belongs, gel electrophoresis will be performed. In preparation for gel electrophoresis, polymerase chain reaction (PCR) will be performed and a 2% agarose gel will be made.

Procedure:

1. Once an aseptic zone was established, 12.5 µL of 1X MM, 2 µL of extracted DNA, and 6.5 µL of ddH₂O were combined three times into three different microcentrifuge tubes.
2. 4 µL of primer 1, primer 2, and primer 3 were placed into their correlating microcentrifuge tubes.
3. Placed microcentrifuge tubes in thermo-cycler and activated program STU.
4. 40 mL of 1X TAE and 0.8 g of powdered agarose were combined and swirled together in an Erlenmeyer flask.
5. Heated the Erlenmeyer flask until the mixture was boiling then mixed solution until the bubbles disappeared. Repeated until the solution was consistent.
6. Allowed the solution to cool until it was cool enough to touch.
7. Added 2.0 µL of EtBr to the flask to achieve a concentration of 0.5 µg/µL.
8. Poured mixture into gel apparatus and placed comb.
9. Once the gel solidified, the comb was removed, and TAE buffer was poured over the gel to keep it from drying out.

Observations:

• The thermocycler used the program STU which started with 5 minutes of initial denaturation at 98.0ºC. Then, 35 cycles of 30 seconds at 98.0ºC followed by 30 seconds at 55.0ºC followed by 45 seconds at 72.0ºC occurred. After the cycles, a final extension happened at 72.0ºC for 5 minutes.
• Unfortunately, it was realized later that DNA concentration was not taken into account when making PCR Mix.
• Also, it was realized later that no controls were made.
• The gel form had 8 wells and had a hazy white tint to it. The following picture shows the gel created.

Next Steps:

Perform gel electrophoresis with DNA samples on agarose gel.