Prbbbb:vector pcr: Difference between revisions
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# add 1µl DpnI, incubate for 1h @ 37°C | # add 1µl DpnI, incubate for 1h @ 37°C | ||
# proceed with [Phosphatase_treatment_of_linearized_vector phosphatase treatment] | # proceed with [[Phosphatase_treatment_of_linearized_vector phosphatase treatment]] | ||
# purify with PCR purification kit | # purify with PCR purification kit | ||
#*elute in water **not** elution buffer | #*elute in water **not** elution buffer |
Revision as of 04:18, 21 March 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 escape 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 (CRG) (fusion format, ACCGGTTAATACTAGTAGCGGCC)
- reverse suffix primer rg0302 (CRG) (fusion format, GCCGGCCATCTAGAAGCG)
- vector template DNA
Procedure
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;
- 35x (10"@98°C; 15"@68C; 1'30"@72C);
- 10'@72C;
- inf. 4C
Post-Processing
- add 1µl DpnI, incubate for 1h @ 37°C
- proceed with Phosphatase_treatment_of_linearized_vector phosphatase treatment
- 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.
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References
Example reference
Contact
or instead, discuss this protocol.