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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Summary)
(Summary)
(2 intermediate revisions not shown.)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Summary==
+
==2/9/2013==
* Type IIS assembly - PCR products
* Type IIS assembly - PCR products
Line 30: Line 30:
| 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. BL02 = 1431<br> 4. BL03 = 1731<br> 5. BL04 = 1686<br> 6.fshPCD = 186<br>
+
| 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
| 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]
| 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]
|-
|-
Line 52: Line 52:
-
Purification (Completed on 1-25-2013)
+
Purification (Completed on 2-09-2013)
-
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O  
+
* Clean with Zymo DNA clean &concentrator kit; eluted w/ 20 μL dH<sub>2</sub>O  
* Measure [DNA] (1-29-2013)
* Measure [DNA] (1-29-2013)

Revision as of 18:43, 14 February 2013

Engineering PC-TFs Main project page
Previous entry      Next entry

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



Personal tools