User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10: Difference between revisions
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== | ==01/10/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Violacein: successful growth; re-streak | ||
* | * Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13 | ||
* Golden gate trial 2: new PCR primers | |||
---- | ---- | ||
''' | '''Violacein'''<br> | ||
* | * Plate 1: Kan plasmid | ||
* Plate 2: Amp plasmid (violacein exp.) | |||
---- | |||
'''Golden gate trial 1''' | |||
* 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. | |||
---- | |||
'''Golden gate trial 2''' | |||
* Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1. | | 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) || 2 | | DNA(plasmid) || 0.2 μL | ||
|- | |- | ||
| | | primer 1 || 1.0 | ||
|- | |- | ||
| | | primer 2 || 1.0 | ||
|- | |- | ||
| | | 2x GoTaq || 25.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 22.8 | ||
|- | |- | ||
| || | | || 50.0 μL | ||
|} | |} | ||
PCR | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
* | Purification | ||
* | * Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O | ||
* Measure [DNA] | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Sample | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | OD 260 | ||
| | | bgcolor=#cfcfcf | 260/280 | ||
| | | bgcolor=#cfcfcf | ng/μL | ||
| | |||
|- | |- | ||
| | | 1. pSB1A3 || 0.126 || 1.888 || 77.357 | ||
|- | |- | ||
| | | 2. hPCD || 0.069 || 2.192 || 17.025 | ||
|- | |- | ||
| | | 3. BL01 || 0.121 || 1.898 || 66.878 | ||
|} | |} | ||
Dilutions: to final concentration of 20 fmol/μL | |||
{| {{table}} cellspacing="3" <!-- | {| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | ||
|- bgcolor=#cfcfcf | |- 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 --> | |||
{| {{table}} cellspacing="3" <!-- | |- valign="top" | ||
|- | | Reagent || 1 || 2 | ||
| | |||
|- | |- | ||
| 1. | | 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 | | 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
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01/10/13
Violacein
Golden gate trial 1
Golden gate trial 2
PCR
Thermal cycling
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