Prbbbb:fusion biobrick construction v1: Difference between revisions
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| Line 21: | Line 21: | ||
useful for primer design: | useful for primer design: | ||
* [http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] | * [http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] -- doesn't offer all salt parameters | ||
* [https://www.finnzymes.fi/tm_determination_old.html Phusion annealing Temperature calculator] | * [https://www.finnzymes.fi/tm_determination_old.html Phusion annealing Temperature calculator] | ||
* CLC workbench has a very nice primer design tool | |||
# design primers to 5' (left) and 3' (right) end of your Biobrick insert using these parameters: | # design primers to 5' (left) and 3' (right) end of your Biobrick insert using these parameters: | ||
#* desired annealing Temperature: 60-62°C | #* desired annealing Temperature: 60-62°C | ||
#* method: nearest | #* method: nearest neighbor | ||
#* [primer]: 500 nM | #* [primer]: 500 nM | ||
#* [salt]: 50 nM | #* [salt]: 50 nM | ||
#* [MgCl2]: 1.5 mM | #* [MgCl2]: 1.5 mM | ||
#* [dNTP]: 200 nM | #* [dNTP]: 200 nM | ||
# Add the following sequence to beginning of forward primer: | |||
<code>ctt cta gat ggc cgg c</code> | |||
# Add the following sequence to beginning of reverse primer: | |||
<code>ccg cta cta gta tta acc ggt</code> | |||
# note fw and reverse primer sequence (reverse complement) | |||
''Under construction!!'' | ''Under construction!!'' | ||
Revision as of 04:20, 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
useful for primer design:
- OligoCalc -- doesn't offer all salt parameters
- Phusion annealing Temperature calculator
- CLC workbench has a very nice primer design tool
- 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
- 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
- note fw and reverse primer sequence (reverse complement)
Under construction!!
setup PCR reaction
| µl, 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;
- 25x (10"@98°C; 15"@60C; 1'30"@72C);
- 10'@72C;
- inf. 4C
Post-Processing
- add 1µl DpnI, incubate for 1h @ 37°C
- purify with PCR purification kit
- elute in water **not** elution buffer
- dilute to standard concentration: 50ng/µl
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.
