Part:BBa_K5087014

EcoF

Introduction

This forward primer was designed to incorporate the EcoRI restriction site into the SynLOCK Cassette (BBa_K5087017). Our objective was to amplify the entire cassette for transfer between different backbones using BioBrick assembly. To achieve this, we needed to introduce EcoRI and SpeI sites flanking the cassette, which was the focus of our PCR reaction.

Experimental Validation

PCR Setup

For this PCR, we selected a 340 ng/μl sample of the plasmid containing the SynLOCK Cassette, known as the SVR plasmid (for more information, you can visit the BBa_K5087017 part site). It was diluted by taking 2 μl of the sample and adding 48 μl of water.

Two reactions were prepared in a final volume of 25 µl.

Table 1: PCR Reaction Master Mix components (for a single reaction)

Component	Volume [µl]
Q5 mix	12.5
EcoF primer (BBa_K5087014) (10 µM)	1.25
SpeR primer(BBa_K5087015) (10 µM)	1.25
H₂O	9.0
Total	24

After pipetting the master mix into PCR tubes, 1 µl of the SVR plasmid dilution was added to all samples except for the negative control (which was substituted with H₂O).

Thermocycler Program:

• Initial denaturation: 98°C for 30s
• Denaturation: 98°C for 10s
• Annealing: 70°C (for negative control and one sample), 65°C (for another sample) for 15s
• Extension: 72°C for 25s
• Final extension: 72°C for 2min
• Hold: 4°C indefinitely

Steps 2, 3, and 4 were looped 30 times.

Note: The annealing temperature was calculated using the NEB Tm Calculator tool and was estimated at 72°C. The negative control was performed at 70°C.

Figure 1. Gel electrophoresis of the SynLOCK Cassette amplified with EcoF and SpeR primers.

Conclusions:

Expected bands of 1045 bp were obtained for the PCR reaction product, proving the efficiency of both used primers.

Sequence

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 5
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5
  Illegal NotI site found at 11
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 5
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 5
  Illegal XbaI site found at 20
  
• 1000
  COMPATIBLE WITH RFC[1000]