User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10
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'''Golden gate trial 1''' | '''Golden gate trial 1''' | ||
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar | * Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar | ||
| + | * Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures. | ||
| Line 50: | Line 51: | ||
PCR | PCR | ||
* 95°C, 3 min. | * 95°C, 3 min. | ||
| - | * [95°C, | + | * [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35 |
* 72°C, 3 min. | * 72°C, 3 min. | ||
* 4°C, ∞ | * 4°C, ∞ | ||
| Line 73: | Line 74: | ||
| - | Dilutions | + | Dilutions: to final concentration of 20 fmol/μL |
{| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | {| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | ||
|- valign="top" | |- valign="top" | ||
| Line 90: | Line 91: | ||
| - | + | Golden Gate Reactions | |
# hPCD + BL01 + pSB1A3 | # hPCD + BL01 + pSB1A3 | ||
# pSB1A3 | # pSB1A3 | ||
| Line 115: | Line 116: | ||
|} | |} | ||
| - | Thermal | + | Thermal cycling |
* [45°C, 2 min.; 16°C, 5 min.] x25 | * [45°C, 2 min.; 16°C, 5 min.] x25 | ||
* 60°C, 20 min. | * 60°C, 20 min. | ||
* 80°C, 20 min. | * 80°C, 20 min. | ||
| + | * 4°C, ∞ | ||
Current revision
 Karmella's BioBrick Cloning
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01/10/13
Violacein
Golden gate trial 1
Golden gate trial 2
PCR
Thermal cycling
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