Team:SCU-China/Linkage

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Revision as of 22:16, 16 October 2014

Linkage Protocol

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According to the advice of IGEM Competition, there are two linkage methods that we use in experiment. The first one is “3A” Assembly and the second is “Traditional” Assembly.

For “3A” Assembly, we set up the following system (20 μl):
Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)	About 100 ng
Insert DNA 1	The molar ratio of Insert DNA to Backbone is 3:1 to 5:1
Insert DNA 2	Same to Insert DNA 1
10 x T4 DNA Ligase Buffer	2 μl
T4 DNA Ligase	1 μl
H2O	Up to 20 μl

For “Traditional” Assembly, we set up the following system (20 μl):

Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)	About 100 ng
Insert DNA	The molar ratio of Insert DNA to Backbone is 3:1 to 5:1
10 x T4 DNA Ligase Buffer	2 μl
T4 DNA Ligase	1 μl
H2O	Up to 20 μl

For constructing the Biobricks that we want to submit, we set up the PCR protocol and PCR system as bellows:

PCR system (For 50μL)
Materials	Volume
Taq DNA polymerase	0.5μL
Template	0.5μL
Forward Primer	1μL
Reverse Primer	1μL
dNTPs (2.5mM)	4μL
10x Buffer (Mg2+ free)	5μL
MgCl2 (25mM)	3μL
Sterilized diluted water	35μL

Note: The content of template should be less than 500ng for the 50μL system. For all of our experiments, the content of template of 0.5μL DNA lower than 500ng.

PCR protocol
Temperature	Times
95℃	5min.	30-35 Times
95℃	30sec.
55-65℃	30sec.
72℃	1-2min.
72℃	10min.

Note: for finding the optimal annealing temperature, we have done a series of gradient of annealing temperature first. And as for the elongation time, Taq DNA polymerase could synthesize DNA as the speed of 1000-2000 nucleotides per minute.

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-  <p></p><p>According to the advice of IGEM Competition, there are two linkage methods that we use in experiment. The first one is &#8220;3A&#8221; Assembly and the second is &#8220;Traditional&#8221; Assembly.</p><p>For &#8220;3A&#8221; Assembly, we set up the following system (20 &#956;l):</p><table><tr><td><p>Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)</p>
+  <p>According to the advice of IGEM Competition, there are two linkage methods that we use in experiment. The first one is &#8220;3A&#8221; Assembly and the second is &#8220;Traditional&#8221; Assembly.</p><table class="table table-striped"> <caption>For &#8220;3A&#8221; Assembly, we set up the following system (20 &#956;l):</caption><tr><td><p>Linear Backbones from IGEM Competition( with K+, C+, A+, or T+)</p>
 </td><td><p>About 100 ng</p>
 </td>