Part:BBa_K4734002

Reverse Primer for Linearizing Vector

Usage and Biology

The primer that we designed is meant to linearize the M13KE Phage. We will need to place the primer at the exact spot, so that it can linearize the vector in order for us to insert our chosen epitope into the sequence. The primer is placed in the middle of the M13KE phage and the reverse and forward primers are flipped, so that it can linearize the prime in accordance with each other and the 5' and 3'. We followed a PCR amplification protocol in order to linearize the vector. The following protocol is listed below:

Materials - Thin-walled 200 uL PCR tubes -2 mL Eppendorf tube

5X Phusion HF Buffer - 10 μL 10 mM dNTPs - 1 μL Template DNA - 2.5 μL Klenow Polymerase Fragment - 0.5 μL Sterile water - 26.5 μL 10 μM Forward Primer (1 & 2) - 1 μL 10 μM Reverse Primer (1 & 2) - 1 μL

Procedure: 1. In the Eppendorf tube, add all reagents listed in the table except for the primers. This will be your Master Mix. 2. Add 18 µL of the Master Mix, 1 µL of 10 μM Forward Primer 1, and 1 µL 10 μM Reverse Primer 1 to a PCR tube. Label this tube PCR 1. 3. Add 18 µL of the Master Mix, 1 µL of 10 μM Forward Primer 2, and 1 µL 10 μM Reverse Primer 2 to a PCR tube. Label this tube PCR 2. 4. Place the PCR 1 tube into the selected thermocycler. 5. Once the lid to the thermocycler is properly closed, start the required amplification program, as in Table 2.

Initial Denaturation - 98 C - 30 seconds - 1 Denaturation - 98 C - 10 seconds - 35 Annealing - 55 C - 30 seconds- 35 Extension - 72 C - 3 minutes 42 seconds - 35 Final Extension - 72 C - 10 minutes - 1 Hold - 10 C - ∞ - 1

6. Place the PCR 2 tube into the selected thermocycler. 7. Once the lid to the thermocycler is properly closed, start the required amplification program, as in Table 3.

These were our DNA Gel Results from the Amplification:

The vector should be linearized and amplified if the protocol is followed exactly. You can now use this linearized plasmid to perform a phage display and ligation protocol in order to insert your chosen or designed epitope into the sequence so that it may enter the lymph to produce an immune response.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]