Prbbbb:vector pcr: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 16: | Line 16: | ||
*vector template DNA | *vector template DNA | ||
**pSB1AC3F -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18901] | **pSB1AC3F -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18901] | ||
**pSB1AK3F -- | **pSB1AK3F -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18902] | ||
**pSB1AT3F -- | **pSB1AT3F -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18903] | ||
==Procedure== | ==Procedure== |
Revision as of 08:10, 4 February 2009
Overview
A PCR reaction to generate linearized construction vector backbones for Fusion(Freiburg)-formatted Biobrick assemblies.
The classic assembly protocol digests the target vector (usually pSB1*) in parallel to the digestions of the two Biobrick parts. We prefer to create a stock of target backbone by PCR with a fast high-fidelity polymerase. This way, mutations in the ccdB death cassette of the target vector cannot circumvent selection.
Materials
- 100 ul + PCR tubes
- Phusion HotStart Polymerase 2 U/ul
- Phusion HF Buffer 5x
- dNTP mix 10mM each nucleotide
- ddH2O
- reverse prefix primer rg0301 (fusion format)
- reverse suffix primer rg0302 (fusion format)
- vector template DNA
- pSB1AC3F -- BBa_J18901
- pSB1AK3F -- BBa_J18902
- pSB1AT3F -- BBa_J18903
Procedure
setup PCR reaction
µl, single reaction | 3.5xMaster | 10xMaster | |
---|---|---|---|
H2O | 76µl | 266 | 760 |
5x HF Buffer | 20µl | 70 | 200 |
10mM dNTP | 2µl | 70 | 200 |
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;
- 35x (10"@98°C; 15"@68C; 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.