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

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==Summary==
==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:
 
# template, primer 1, primer 2
# template, primer 1, primer 2
# template, primer 1, primer 2
# template, primer 1, primer 2
 
 
{| {{table}} border="1" cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
| 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]
|-
| DNA(plasmid) || 0.2 μL
|-
| primer 1 || 1.0
|-
| primer 2 || 1.0
|-
| 2x GoTaq || 25.0
|-
| dH<sub>2</sub>O || 22.8
|-
| &nbsp; || 50.0 μL
|}
 
PCR
* 95°C, 3 min.
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
* 72°C, 3 min.
* 4°C, ∞
 




Revision as of 13:38, 25 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. template, primer 1, primer 2
  2. template, primer 1, primer 2
  3. template, primer 1, primer 2
  4. template, primer 1, primer 2


Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520 		Hover name
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

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞



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