Prbbbb:fusion biobrick construction v1: Difference between revisions
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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. | 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: HotStart Phusion 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. | |||
<table> | <table> |
Revision as of 15:49, 17 February 2009
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
- 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
- Add the following sequence to beginning of forward primer:
ctt cta gat ggc cgg c
- Add the following sequence to beginning of reverse primer:
ccg cta cta gta tta acc ggt
- 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: HotStart Phusion 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.
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Post-Processing
- add 1µl DpnI, incubate for 1h @ 37°C
- desalt and purify with PCR purification kit
In-Fusion reaction
Follow standard Infusion protocol:
- mix vector and insert DNA in molar ratio of 1:2 into 10µl ddH2O
- add DNA mix to Dry-Down Infusion tube
- let stand for a minute
- carefully pipette up & down until dry-down mix is disolved
- put tubes into PCR device and run:
- stop reaction with 30µl 10mM TE Buffer
Transformation
Follow the standard transformation protocol.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Example reference
Contact
or instead, discuss this protocol.