36
edits
SPLiCE: Difference between revisions
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- 25ul of 2X T4 ligase buffer (66mM Tris-HCL. 10mM MgCl2, 1mM Dithiothreitol, 1mM ATP, 7.5% Polyethylene glycol (PEG6000), pH 7.6 @ 25°C) <br> | - 25ul of 2X T4 ligase buffer (66mM Tris-HCL. 10mM MgCl2, 1mM Dithiothreitol, 1mM ATP, 7.5% Polyethylene glycol (PEG6000), pH 7.6 @ 25°C) <br> | ||
30 minutes at 37°C<br> | 30 minutes at 37°C<br> | ||
Quick SPLiCE (10ul or 50ul reaction): | Quick SPLiCE (10ul or 50ul reaction): <br> | ||
- 50-200ng linear vector <br> | - 50-200ng linear vector <br> | ||
- Appropriate amount of insert DNA in a 3:1 molar ratio of insert to vector <br> | - Appropriate amount of insert DNA in a 3:1 molar ratio of insert to vector <br> | ||
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Parts used<br> | Parts used<br> | ||
For all our reactions we used the following parts <br> | For all our reactions we used the following parts <br> | ||
- Insert BBa_J04450 amplified from the 2014 distribution part, originally in pSB1K3 backbone ( | - Insert BBa_J04450 amplified from the 2014 distribution part, originally in pSB1K3 backbone (50ng/ul)<br> | ||
Amplified with the following primers FWD: 5' GTTCTTCGAATTCGCGGCCGCTTCTAGAGAAAGAGGAGAAATACTAGATGGCTTCC 3' REV: 5' GACTGCAGCGGCCGCTACTAGTA 3' <br> | Amplified with the following primers FWD: 5' GTTCTTCGAATTCGCGGCCGCTTCTAGAGAAAGAGGAGAAATACTAGATGGCTTCC 3' REV: 5' GACTGCAGCGGCCGCTACTAGTA 3' <br> | ||
- Linear pSB1C3 backbone amplified from BBa_K554001 including the biobrick prefix and suffix ( | - Linear pSB1C3 backbone amplified from BBa_K554001 including the biobrick prefix and suffix (100ng/ul)<br> | ||
Amplified with the following primer FWD: 5'TACTAGTAGCGGCCGCTGCAGTCCG3' REV: 5'CTCTAGAAGCGGCCGCGAATTCCAG3' | Amplified with the following primer FWD: 5'TACTAGTAGCGGCCGCTGCAGTCCG3' REV: 5'CTCTAGAAGCGGCCGCGAATTCCAG3' | ||
