Haynes Lab:Notebook/Engineering PC-TFs/2013/01/25: Difference between revisions
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== | ==01/25/13== | ||
* | * Type IIS assembly - day 1 | ||
---- | |||
PCR | |||
* Resuspend new primers to a final concentration of 100 μM | |||
* Make 10 μM dilutions in separate tubes | |||
* All of the reactions are as follows: | |||
# pSB1A3, gg0001, gg0002 | |||
# hPCD, gg0003, gg0004 | |||
# BL02, gg0005, gg0007 | |||
# BL03, gg0005, gg0008 | |||
# BL04, gg0005, gg0009 | |||
# fshPCD, gg0010, gg0012 | |||
{| {{table}} border="1" cellspacing="3" <!-- PCR rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL02 = 1431<br> 4. BL03 = 1731<br> 5. BL04 = 1686<br> 6.fshPCD = 186<br> | |||
| rowspan="7" | [[Image:gg_gel1-25-2013.jpg|200px|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA(plasmid) || 0.2 μL | |||
|- | |||
| primer 1 || 1.0 | |||
|- | |||
| primer 2 || 1.0 | |||
|- | |||
| 2x GoTaq || 25.0 | |||
|- | |||
| dH<sub>2</sub>O || 22.8 | |||
|- | |||
| || 50.0 μL | |||
|} | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
Purification (Completed on 1-25-2013) | |||
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O | |||
* Measure [DNA] (1-29-2013) | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Sample | |||
| bgcolor=#cfcfcf | OD 260 | |||
| bgcolor=#cfcfcf | 260/280 | |||
| bgcolor=#cfcfcf | ng/μL | |||
|- | |||
| 1. pSB1A3 || .084|| 1.801 || 83.954 | |||
|- | |||
| 2. hPCD || .017 || 1.734|| 17.344 | |||
|- | |||
| 3. BL02 || .058 || 1.764|| 58.413 | |||
|- | |||
| 4. BL03 || .082 || 1.831|| 81.877 | |||
|- | |||
| 5. BL04 || .05 || 1.888 || 49.561 | |||
|- | |||
| 6. fshPCD || .021 || 1.622 || 21.378 | |||
|} | |||
Revision as of 18:31, 6 September 2013
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01/25/13
PCR
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