Registry of standard biological parts/PCR-based assembly/Protocol

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Contents

Plan Construction

As an example we will be building:

  1. <bbpart>J04500</bbpart>+<bbpart>I13501</bbpart> = <bbpart>J04450</bbpart> (RFP expression device)
    • Promoter/RBS + FP

Possibly try this later:

  1. <bbpart>R0010</bbpart>+<bbpart>I13504</bbpart> = <bbpart>J04430</bbpart> (GFP expression device)
    • Promoter + RBS/FP

Primer Design

External Primers

  1. Design 20bp primers from the ends of the finished assembly.
  2. Add 15bp overhangs based on digesting 1AK3 with E/P using this primer design tool for primers for the In-Fusion cloning method

Fusion primers

PCR Reactions

PCR Reaction A (PCR the individual parts)

PCR mix

Component Volume/50uL rxn
H2O 24.5
5x Phusion HF Buffer 10uL
2.5mM dNTPs 4uL
primer A (5uM) 5uL
primer B (5uM) 5uL
template DNA 1uL
Phusion DNA polymerase 0.5uL
PCR A: successful PCR reaction for annealing temp 60-72C
PCR A: successful PCR reaction for annealing temp 60-72C

PCR conditions

  • Used the same conditions for J04500 and I13501
  1. Initial Denaturation – 98C for 30s
  2. Denaturation – 98C for 5s
  3. Annealing – 65C for 15s @ Tm+3C
  4. Extension – 72C 13s
  5. repeat to (2) 20 times.
  6. Final extension – 72C for 5-10min
  7. 4C indef

PCR Reaction B

  • Going to try this 2 ways:
    1. Dilute the PCR produces of PCR A 1/1000 (add 1uL of each as the template) and then add them together with external primers
    2. Do extension with no primers and add the undiluted PCRA as primers (e.g. 5uL of each one with no template added).

PCR mix #1

Component Volume/50uL rxn
H2O 23.5
5x Phusion HF Buffer 10uL
2.5mM dNTPs 4uL
primer A (5uM) - J04500_fus_FWD2 5uL
primer B (5uM) - I13501_fus_REV2 5uL
template DNA (1uL of 1/1000 dilution of each product from PCR A) 2uL
Phusion DNA polymerase 0.5uL
PCR B: The no primers (lane 4) looked good
PCR B: The no primers (lane 4) looked good

PCR mix #2

Component Volume/50uL rxn
H2O 24.5
5x Phusion HF Buffer 10uL
2.5mM dNTPs 4uL
primer A (5uM) - J04500 PCR A product undiluted 5uL
primer B (5uM) - I13501 PCR A product undiluted 5uL
Phusion DNA polymerase 0.5uL

References

Sauer:Stitching Genes by PCR

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