November 14, 2012
- The DNA was extracted from the gel slices using the Zymoclean Gel DNA Recovery Kit
| Sample | 260 | 280 | 260/280 | ng/μL
|
| H2B | 0.006 | 0.004 | 1.463 | 6.384
|
| LOV | 0.011 | 0.004 | 2.769 | 11.492
|
| pSB1A3 | 0.004 | 0.001 | 2.857 | 4.233
|
- ---Karmella 21:34, 15 November 2012 (EST): Next step - ligation & transformation
Refer to this page for a full description: http://openwetware.org/wiki/Haynes:Assembly101
- Use 20 ng of vector for each ligation = 5 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
| | H2B | LOV | Negative Control
|
| Insert DNA (X ng) | 1.0 μL | 1.0 | 0
|
| Vector DNA (50 ng) | 5.0 | 5.0 | 5.0
|
| 2x Roche Rapid Ligation buffer | 7.0 | 7.0 | 7.0
|
| New England Biolabs T4 ligase | 1.0 | 1.0 | 1.0
|
| dH2O | 0 | 0 | 1.0
|
| TOTAL | 14.0 | 14.0 | 14.0
|
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 10 minutes.
|
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
|
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