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 (2x mol vector) |
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
|