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

Under construction!!

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"@98C
5x	(10"@98°C; 15"@63C; 1'@72C);
25x	(10"@98°C; 1'@72C);
	10'@72C;
	inf. 4C

Post-Processing

1. add 1µl DpnI, incubate for 1h @ 37°C
2. 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.