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November 20, 2012
- Made liquid cultures of pSB1A3 and PT7CFE1
- In a 30 μL reaction, cut a pSB1A2 plasmid (from Rene) XbaI and SpeI, then ran on 1% gel and used Zymoclean Gel DNA Recovery Kit
Reagent |
pSB1A2
|
DNA |
15.0
|
XbaI |
1.0
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SpeI |
1.0
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10x buffer |
3.0
|
dH2O |
10.0
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Total vol. |
30.0
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Results of the enzyme digest is shown, with both pSB1A3 bands on the right. The gel was loaded with 30 μL samples. The bands that were cut are outlined in green.
- Results from gel recovery
Sample |
260 |
280 |
260/280 |
ng/μL
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pSB1A3 |
0.017 |
0.008 |
2.132 |
17.238
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Refer to this page for a full description: http://openwetware.org/wiki/Haynes:Assembly101
- Use 20 ng of vector for each ligation = 1 μL pSB1A3
- Use 2x moles of H2B insert, relative to vector: xμL H2B insert = 2 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 6.384 ng/μL H2B = 1.28 μL H2B
- Use 2x moles of LOV insert, relative to vector: xμL LOV insert = 2 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 11.492 ng/μL LOV = 0.8 μL LOV
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H2B |
LOV |
Negative Control
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Insert DNA (2x mol vector) |
1.0 μL |
1.0 |
0
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Vector DNA (50 ng) |
1.0 |
1.0 |
1.0
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2x Roche Rapid Ligation buffer |
7.0 |
7.0 |
7.0
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New England Biolabs T4 ligase |
1.0 |
1.0 |
1.0
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dH2O |
4.0 |
4.0 |
5.0
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TOTAL |
14.0 |
14.0 |
14.0
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Mix the reaction(s) thoroughly by flicking the tube. Incubate at room temperature for 10 minutes.
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Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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