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, which saves time and makes this protocol rather reliable.

The same protocol can be applied to other Biobrick formats -- simply replace the primer flanks by the 16 inner-most bases of your favorite prefix and suffix sequence.

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
I Primer design


 * 1) design primers to 5' (left) and 3' (right) end of your Biobrick insert using these parameters:
 * 2) * desired annealing Temperature: 60-62°C
 * 3) * method: nearest neighbor
 * 4) * [primer]: 500 nM; [salt]: 50 nM; [MgCl2]: 1.5 mM; [dNTP]: 200 nM
 * 5) * try ending with one or two G/C at the 3' terminal
 * 6) Add the following sequence to the beginning of the forward primer:
 * 7) *Fusion (Freiburg) format:  
 * 8) Add the following sequence to the beginning of the reverse primer (i.e. the reverse complement of the end of your insert):
 * 9) *Fusion (Freiburg) format:  
 * 10) 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

II 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.


 * extension time text = (kb insert length) × 25"
 * annealing temperature Ta = (primer annealing) + 3°C

III Post-Processing


 * 1) add 1µl DpnI, mix well, incubate for 1h @ 37&deg;C, 20' @ 80&deg;C
 * 2) verify PCR result on an agarose gel
 * 3) desalt and purify with PCR purification kit

IV 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 40µl 10mM TE Buffer

Note about In-Fusion efficiency:
 * the In-Fusion mix has toxicity issues, in particular with Top10 cells
 * dilute reaction to 100µl when using Top10
 * or use DH5alpha super-competent cells
 * using lower volumes for the transformation increases the colony count

V Transformation

Follow the standard transformation protocol: use 2.5µl diluted reaction for transformation of 50µl competent cells.

Note: Standard Top10 cells show rather low transformation efficiencies with In-Fusion products. We have had good experiences with Invitrogen MaxEfficiency DH5alpha cells and others report good results with Clonetech FusionBlue competent cells. Simply diluting (see above) or reducing the amount of reaction may also help.

VI 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.

Contact

 * Raik

or instead, discuss this protocol.