Difference between revisions of "Part:BBa K1789013"

(→‎Enzyme digestion test)
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==Experimental Validation==
 
==Experimental Validation==
  
−
===Sequencing===
+
This part is validated through four ways: Enzyme cutting, PCR, and Sequence
 +
===PCR===
  
−
This part is sequenced as correct after construction.
+
'''Methods'''
  
−
===Enzyme digestion test ===
+
The PCR is performed with Premix EX Taq by Takara.
  
−
A double-enzyme digestion test was implemented to verify this part.
+
The PCR protocol is selected based on the Users Manuel.
−
 
+
The Electrophoresis was performed on a 1％ Agarose glu.
−
[[File:SCAF1_Ter酶切.jpg|500px|]]
+
  
−
The length of the digested DNA fits the design.
+
'''Results'''
  
 +
[[File:1.jpg|150px|]]
 +
 +
The result of the agarose electrophoresis was shown on the picture above.
 +
 +
===Enzyme cutting===
 +
 +
'''Methods'''
 +
 +
After the assembly ,the plasmid was transferred into the Competent ''E. coli'' DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting.
 +
The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from ''TIANGEN''. The cutting procedure was performed with EcoRI and XbaI restriction endonuclease bought from ''TAKARA''.
 +
 +
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours.
 +
The Electrophoresis was performed on a 1％ Agarose glu.
 +
 +
'''Results'''
 +
 +
[[File:SCAF1_Ter酶切.jpg|150px|]]
 +
 +
The result of the agarose electrophoresis was shown on the picture above.
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1789013 parameters</partinfo>
 
<partinfo>BBa_K1789013 parameters</partinfo>

Revision as of 15:09, 18 September 2015

SCAF1+Ter

This is a combination of scaffold and termination.


Usage and Biology

Usage: This part is scaf1 which is associated with a terminator. Scaf1 is used to bind TALE1 and TALE3 protein to make them fixed on the DNA. We create this part to make it more convenient every time we are required to add a scaffold.


Biology: Scaf1 is a DNA scaffold which can bind TALE protein specifically to achieve the fixing of protein or enzyme. This part is scaf1 with a terminator after it.

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 145
    Illegal AgeI site found at 175
    Illegal AgeI site found at 205
    Illegal AgeI site found at 235
    Illegal AgeI site found at 265
    Illegal AgeI site found at 295
    Illegal AgeI site found at 325
  • 1000
    COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is validated through four ways: Enzyme cutting, PCR, and Sequence

PCR

Methods

The PCR is performed with Premix EX Taq by Takara.

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu.

Results

1.jpg

The result of the agarose electrophoresis was shown on the picture above.

Enzyme cutting

Methods

After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with EcoRI and XbaI restriction endonuclease bought from TAKARA.

The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Results

SCAF1 Ter酶切.jpg

The result of the agarose electrophoresis was shown on the picture above.

Functional Parameters

CHIp

Enzyme digestion test /