Haynes Lab:Notebook/Engineering PC-TFs/2013/02/09

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(Summary)
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==Summary==
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==2/9/2013==
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*  
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* Type IIS assembly - PCR products
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----
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PCR
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* Resuspend new primers to a final concentration of 100 μM
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* Make 10 μM dilutions in separate tubes
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* All of the reactions are as follows:
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# pSB1A3, gg0001, gg0002
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# hPCD, gg0003, gg0004
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# BL02, gg0005, gg0007
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# BL03, gg0005, gg0008
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# BL04, gg0005, gg0009
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{| {{table}} border="1" cellspacing="3" <!-- PCR rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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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
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| rowspan="7" | [[Image:gg_gel2-9-2013.jpg|200px|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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|-
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| DNA(plasmid) || 0.2 μL
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|-
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| primer 1 || 1.0
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|-
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| primer 2 || 1.0
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|-
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| 2x GoTaq || 25.0
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|-
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| dH<sub>2</sub>O || 22.8
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|-
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| &nbsp; || 50.0 μL
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|}
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* 95°C, 3 min.
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* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
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* 72°C, 3 min.
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* 4°C, ∞
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Purification (Completed on 2-09-2013)
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* Clean with Zymo DNA clean &concentrator kit; eluted w/ 20 μL dH<sub>2</sub>O
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* 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 || .079|| 1.837 || 79.379
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|-
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| 2. hPCD || .022 || 1.883|| 22.239
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|-
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| 3. BL02 || .129|| 1.883|| 128.889
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|-
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| 4. BL03 || .079 || 1.918|| 78.72
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|-
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| 5. BL04 || .06 || 1.879 || 59.912
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|}
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Revision as of 19:43, 14 February 2013

Engineering PC-TFs Main project page
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2/9/2013

  • Type IIS assembly - PCR products



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


Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL02 = 1431
4. BL03 = 1731
5. BL04 = 1686
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 2-09-2013)

  • Clean with Zymo DNA clean &concentrator kit; eluted w/ 20 μL dH2O
  • Measure [DNA] (1-29-2013)
Sample OD 260 260/280 ng/μL
1. pSB1A3 .079 1.837 79.379
2. hPCD .022 1.883 22.239
3. BL02 .129 1.883 128.889
4. BL03 .079 1.918 78.72
5. BL04 .06 1.879 59.912



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