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

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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
  
  
Line 27: Line 29:
 
| 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
Line 49: Line 51:
  
  
Purification
+
Purification (Completed on 1-25-2013)
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
+
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O  
* Measure [DNA]
+
 
 +
* Measure [DNA] (1-29-2013)
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
|- valign="top"
 
|- valign="top"
Line 59: Line 62:
 
| bgcolor=#cfcfcf | ng/μL  
 
| bgcolor=#cfcfcf | ng/μL  
 
|-
 
|-
| 1. pSB1A3 || ### || ### || ###
+
| 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
 
|-
 
|-
| 2. ### || ### || ### || ###
+
| 5. BL04 || .05 || 1.888 || 49.561
 
|-
 
|-
| 3. ### || ### || ### || ###
+
| 6. fshPCD || .021 || 1.622 || 21.378
 
|}
 
|}
  

Revision as of 23:52, 31 January 2013

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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