Haynes Lab:Notebook/Engineering PC-TFs/2013/01/25

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(Summary)
(01/25/13)
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* All of the reactions are as follows:
* All of the reactions are as follows:
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# template, primer 1, primer 2
+
# pSB1A3, gg0001, gg0002
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# template, primer 1, primer 2
+
# hPCD, gg0003, gg0004
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# template, primer 1, primer 2
+
# BL02, gg0005, gg0007
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# template, primer 1, primer 2
+
# BL03, gg0005, gg0008
 +
# BL04, gg0005, gg0009
 +
# fshPCD, gg0010, gg0012
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
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| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
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| 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>
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| 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]
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| 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
| DNA(plasmid) || 0.2 μL
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|}
|}
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PCR
 
* 95°C, 3 min.
* 95°C, 3 min.
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
* [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 (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)
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Sample
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| bgcolor=#cfcfcf | OD 260
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| bgcolor=#cfcfcf | 260/280
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| bgcolor=#cfcfcf | ng/μL
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|-
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| 1. pSB1A3 || .084|| 1.801 || 83.954
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|-
 +
| 2. hPCD || .017 || 1.734|| 17.344
 +
|-
 +
| 3. BL02 || .058 || 1.764|| 58.413
 +
|-
 +
| 4. BL03 || .082 || 1.831|| 81.877
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|-
 +
| 5. BL04 || .05 || 1.888 || 49.561
 +
|-
 +
| 6. fshPCD || .021 || 1.622 || 21.378
 +
|}

Revision as of 01:52, 1 February 2013

Engineering PC-TFs Main project page
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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:
  1. pSB1A3, gg0001, gg0002
  2. hPCD, gg0003, gg0004
  3. BL02, gg0005, gg0007
  4. BL03, gg0005, gg0008
  5. BL04, gg0005, gg0009
  6. fshPCD, gg0010, gg0012


Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL02 = 1431
4. BL03 = 1731
5. BL04 = 1686
6.fshPCD = 186
PCR results
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 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 dH2O
  • Measure [DNA] (1-29-2013)
Sample OD 260 260/280 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



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