User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10: Difference between revisions
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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. | |||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520 | | rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520 | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH011013_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(plasmid) || 0.2 μL | | DNA(plasmid) || 0.2 μL | ||
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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, ∞ | ||
Purification | |||
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O | |||
* Measure [DNA] | * Measure [DNA] | ||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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| 3. BL01 || 0.121 || 1.898 || 66.878 | | 3. BL01 || 0.121 || 1.898 || 66.878 | ||
|} | |} | ||
Dilutions: to final concentration of 20 fmol/μL | |||
{| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | DNA | |||
| bgcolor=#cfcfcf | length (bp) | |||
| bgcolor=#cfcfcf | ng/μL | |||
| bgcolor=#cfcfcf | Vol. | |||
| bgcolor=#cfcfcf | + dH<sub>2</sub>O | |||
|- | |||
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3 | |||
|- | |||
| hPCD || 186 || 17.0 || 2.8 || 17.2 | |||
|- | |||
| BL01 || 2520 || 66.9 || 9.8 || 10.2 | |||
|} | |||
Golden Gate Reactions | |||
# hPCD + BL01 + pSB1A3 | |||
# pSB1A3 | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | |||
|- valign="top" | |||
| Reagent || 1 || 2 | |||
|- | |||
| gg2 pSB1A3 || 1.0 || 1.0 | |||
|- | |||
| gg3 hPCD || 1.0 || --- | |||
|- | |||
| gg4 BL01 || 1.0 || --- | |||
|- | |||
| 10x ligase buffer || 1.0 || 1.0 | |||
|- | |||
| NEB T4 lgase || 0.25 || 0.25 | |||
|- | |||
| BsmBI || 0.5 || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 5.25 || 7.25 | |||
|- | |||
| || 10.0 || 10.0 | |||
|} | |||
Thermal cycling | |||
* [45°C, 2 min.; 16°C, 5 min.] x25 | |||
* 60°C, 20 min. | |||
* 80°C, 20 min. | |||
* 4°C, ∞ | |||
Revision as of 21:42, 18 January 2013
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
01/10/13
Violacein
Golden gate trial 1
Golden gate trial 2
PCR
Thermal cycling
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