Difference between revisions of "Part:BBa K530003:Design"

(→‎Design Notes)
(→‎Design Notes)
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This PCR was done with the use of Herculase Enzyme from Agilent.
 
This PCR was done with the use of Herculase Enzyme from Agilent.
  
−
|                       ^ Volume uL ^
+
{|
−
^ Herculase 5X Buffer   | 10         |  
+
|Reagents
−
^ 2.5mM dNTP Mix       | 5         |  
+
|Volume (uL)
−
^ 100-300ng genomic DNA | X         |  
+
|-
−
^ 10uM Forward Primer   | 1.25       |  
+
|Herculase 5X Buffer
−
^ 10uM Reverse Primer   | 1.25       |
+
|10
−
^ Herculase II Enzyme   | 1         |
+
|-
−
^ Sterile Water         | Y          |  
+
|2.5mM dNTP Mix
−
^ Total                 | 50         |
+
|5
 +
|-
 +
|100-300ng DNA
 +
|X
 +
|-
 +
|10uM Forward Primer
 +
|1.25
 +
|-
 +
|10uM Reverse Primer
 +
|1.25
 +
|-
 +
|Herculase II Enzyme
 +
|1
 +
|-
 +
|Sterile Water
 +
|Fill to total
 +
|-
 +
|Total
 +
|50
 +
|}
 +
 
  
 
^ Temperature  ^ Time      ^  Cycles  ^
 
^ Temperature  ^ Time      ^  Cycles  ^

Revision as of 02:48, 22 September 2011

KRE9 Yeast Promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 311
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Promoter was extracted from purified genomic DNA from Saccharomyces Cerevisiae. The PCR reaction performed also served to add the biobrick prefix and suffix to the promoter. PCR and tube contents This PCR was done with the use of Herculase Enzyme from Agilent.

Reagents Volume (uL)
Herculase 5X Buffer 10
2.5mM dNTP Mix 5
100-300ng DNA X
10uM Forward Primer 1.25
10uM Reverse Primer 1.25
Herculase II Enzyme 1
Sterile Water Fill to total
Total 50


^ Temperature ^ Time ^ Cycles ^ | 95 Celsius | 2 minutes | 1 | | 95 Celsius | 20 seconds | 30 | | 55 Celsius | 20 seconds | 30 | | 72 Celsius | 30 seconds | 30 | | 72 Celsius | 3 minutes | 1 |

5ul of the sample were run on a gel with loading dye. The resulting gel confirmed the product was at the right place to be the insert with the biobrick prefix and suffix now attached.

Source

http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=S000003710

References