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From 2011.igem.org

Contents 1 Project 2. 1.1 1.1：The construction of pSB1AC3/PR 1.2 1.2: Gel electrophoresis 1.3 1.3: DNA sequencing 1.4 2.1 The construction of pSG5/PR which is used for eukaryotic expression 1.5 2.3 DNA sequencing 1.6 3.1  The construction of pBABE/PR 1.7 3.2 Gel electrophoresis 1.8 3.3 DNA sequencing 1.9 Notebook

Project 2.

1.1：The construction of pSB1AC3/PR

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PR + pSB1AC3 = pSB1AC3/PR (new part)

Cut PR w/EcoRI & SpeI

Cut pSB1AC3 w/EcoRI & SpeI

Combine the insert and vector with T4 ligase

1.2: Gel electrophoresis

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1: wide range maker 2、3、4、5：the constructed plasmid cutted with SpeI and EcoRI restriction enzymes

 

 

 

 

 

 

 

 

 

 

1.3: DNA sequencing

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The result of DNA sequencing demonstrates that there is no mutation in the new part by comparing to the original sequence.

 

2.1 The construction of pSG5/PR which is used for eukaryotic expression

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PR + pSG5 = pSG5/PR
Cut PR w/EcoRI & BamH I
Cut pSG5 w/EcoRI & BamH I
Combine the insert and vector with T4 ligase

2.3 DNA sequencing

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The result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

3.1  The construction of pBABE/PR

1) PCR for amplifying more insert
Primer 1:         5'-ggg gaa ttc taa acc acc atg ctt gcc-3'
Primer 2:         5'-aaa gaa ttc tca cta tta ggc gtt gct-3'
Reaction system:
10XPCR buffer      5ul
dNTP                    4ul
primer 1                1ul
primer 2                2ul
pfu                     1ul
template               1ul
DMSO                 5ul
ddH2O                31ul
Total                  50ul
Reaction condition:
95℃       5min
95℃       15s
52℃       15s
72℃       2min
72℃       10min
4℃        ∞
2) Ligation

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PR + pBABE = pBABE/PR
Cut PR w/EcoRI
Cut pBABE w/EcoRI
Combine the insert and vector with T4 ligase

3.2 Gel electrophoresis

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1:Middle range maker</a> 　　　 1:DL2,000 DNA Marker

2:cut with EcoR I　　　　　 2:cut with Sal I

Figure 1 indicates that the insert is combined with the vector successfully.
Figure 2 indicates that the insert is in the right direction.

3.3 DNA sequencing

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As before, the result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

 

 

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Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.