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

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# pSB1A3, gg0001, gg0002
# pSB1A3, gg0001, gg0002
# template, primer 1, primer 2
# hPCD, gg0003, gg0004
# template, primer 1, primer 2
# BL02, gg0005, gg0007
# 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  
| 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. BL02 = 1431<br> 4. BL03 = 1731<br> 5. BL04 = 1686<br> 6.fshPCD = 186<br>
| rowspan="7" | [[Image:image.jpg|200px|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| 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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|}
|}


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)
{| {{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 || ### || ### || ###
|-
| 2. ### || ### || ### || ###
|-
| 3. ### || ### || ### || ###
|}




Revision as of 22:35, 27 January 2013

Engineering PC-TFs <html><img src="/images/9/94/Report.png" border="0" /></html> 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 ### ### ###
2. ### ### ### ###
3. ### ### ### ###



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