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Team:UST-Beijing/Notebook
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<h2>3.The experiment</h2> | <h2>3.The experiment</h2> | ||
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<p>fig.1 The key element that affect the RFP are IPTG,Kan and our compound.</p> | <p>fig.1 The key element that affect the RFP are IPTG,Kan and our compound.</p> | ||
<p>https://static.igem.org/mediawiki/2011/1/1d/Quxiantu.jpg" </p> | <p>https://static.igem.org/mediawiki/2011/1/1d/Quxiantu.jpg" </p> | ||
Revision as of 01:59, 28 September 2011
Project I.
1.1：The construction of pSB1AC3（BBa_K603000）/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
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
PR + pSG5 = pSG5/PR
Cut PR w/EcoRI & BamH I
Cut pSG5 w/EcoRI & BamH I
Combine the insert and vector with T4 ligase
2.2 Gel electrophoresis
1: wide range marker
2: the constructed plasmid pSG5/PR cut with EcoR1 and BamH1
2.3 DNA sequencing
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
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95℃ 15s
52℃ 15s
72℃ 2min
25cycles
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72℃ 10min
4℃ ∞
PR + pBABE = pBABE/PR
Cut PR w/EcoRI
Cut pBABE w/EcoRI
Combine the insert and vector with T4 ligase
3.2 Gel electrophoresis
1:Middle range maker 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
As before, the result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.
Project II
1.The construction of lacI-DBD-LXR-LBD
fig.1 Amino acid sequence of lacI-DBD-LXR-LBD chimeric protein,the DNA binding domain of lacI is boxed.
The DNA sequencing(bottom left)and the gel electrophoresis of the digested pET28(bottom right) both demostrate that
the construction of lacI-DBD-LXR-LBD is exactly what we designed before.
2.The gene worknet
3.The experiment
fig.1 The key element that affect the RFP are IPTG,Kan and our compound.
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fig.2 We have tried five compounds,compound B,C,D and E can active the gene and express rfp.The pET28a is
control that have no lacI-DBD-LXR-LBD gene.
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.
