Prbbbb:fusion biobrick construction v1

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Overview

A protocol for creating fusion (Freiburg)-formatted Biobricks from unformatted template DNA.

We use PCR to amplify the insert and to introduce the inner 16 bp of Biobrick prefix and suffix. This insert is then recombined into the linearized vector backbone using Clontech In-Fusion.

Materials

• 100 µl PCR tubes
• Phusion HotStart Polymerase 2 U/ul
• Phusion HF Buffer 5x
• dNTP mix 10mM each nucleotide
• ddH2O
• linear vector backbone DNA from Prbbbb:vector_pcr

Procedure

Primer design

1. design primers to 5' (left) and 3' (right) end of your Biobrick insert using these parameters:
   • desired annealing Temperature: 60-62°C
   • method: nearest neighbor
   • [primer]: 500 nM
   • [salt]: 50 nM
   • [MgCl2]: 1.5 mM
   • [dNTP]: 200 nM
   • try to have one or two G/C at the 3' end
2. Add the following sequence to beginning of forward primer:
   • ctt cta gat ggc cgg c
3. Add the following sequence to beginning of reverse primer:
   • ccg cta cta gta tta acc ggt
4. Order your primers...

useful links for primer design:

   * OligoCalc -- doesn't offer all salt parameters
   * Phusion annealing Temperature calculator
   * CLC workbench has a very nice primer design tool

setup PCR reaction

100µl single reaction 3.5xMaster 10xMaster H2O 76µl 266 760 5x HF Buffer 20µl 70 200 10mM dNTP 2µl 7 20 rg0301 100 µM 0.5µl 1.75 5 rg0302 100 µM 0.5µl 1.75 5 Phusion 1µl 3.5 10 template DNA 0.1µl -- --	PCR Program 30"@98°C 5x (10"@98°C; 15"@Ta; text@72°C); 25x (10"@98°C; 1'@72°C); 10'@72°C ∞ 4°C * extension time text = (insert length in kb) × 25" * annealing temperature Ta = (primer annealing) + 3°C

Post-Processing

1. add 1µl DpnI, incubate for 1h @ 37°C
2. desalt and purify with PCR purification kit
   • elute in water **not** elution buffer
3. dilute to standard concentration: 50ng/µl

Notes

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References

Example reference

1. ISBN:0879697164

Contact

• Raik

or instead, discuss this protocol.