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. That means we avoid any restriction or ligation.

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
• Clontech In-Fusion dry-down kit
• DpnI
• PCR purification kit

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 ending with one or two G/C at the 3' terminal
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

The initial PCR cycles allow the inner part of the primers to anneal. We then switch to two-step PCR since the primers can anneal along their full length to the products of the first rounds.

Note on annealing temperature: The HotStart Phusion enzyme requires annealing temperatures of 60°C or higher. The actual annealing temperature should be 3°C above the lower temperature calculated for any of the two primers. See also the In-Fusion instructions.

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; text@72°C); 10'@72°C ∞ 4°C extension time text = (kb insert length) × 25" annealing temperature Ta = (primer annealing) + 3°C

Post-Processing

1. add 1µl DpnI, mix well, incubate for 1h @ 37°C
2. verify PCR result on an agarose gel
3. desalt and purify with PCR purification kit

In-Fusion reaction

Follow standard Infusion protocol -- add a control with vector-only and one with insert-only DNA:

1. mix vector and insert DNA in molar ratio of 1:2 into 10µl ddH2O
2. add DNA mix to Dry-Down Infusion tube
3. let stand for a minute
4. carefully pipette up & down until dry-down mix is disolved
5. put tubes into PCR device and run:
6. stop reaction with 30µl 10mM TE Buffer

Check the details above against In-Fusion protocol

Transformation

Follow the standard transformation protocol.

Screening

The positive transformation plates should have much more colonies than the control. Screen by colony PCR with the standard BBVF2 and BBVR primers; inoculate positive clones over night for miniprep, restriction test and sequencing.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: This protocol is quick and robust in my hands. There is no need for restriction or ligation steps and, I guess, that's what makes it so reliable.

References

Example reference

1. ISBN:0879697164

Contact

• Raik

or instead, discuss this protocol.