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Team:UST-Beijing/Notebook
From 2011.igem.org
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<p><h1>Project 2.</h1></p> | <p><h1>Project 2.</h1></p> | ||
Revision as of 06:56, 12 September 2011
Contents |
Project 2.
1.1：The construction of pSB1AC3/PR
<img src=" " width="250" height="150"/> |
<img src=" " width="300" height="191" /> |
<img src=" " width="300" height="225"/> |
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
<img src=" " width="400" height="257"/> |
1: wide range maker
|
1.3: DNA sequencing
<img src="
" width="640"/>
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
<img src="
" width="313" height="89" /><img src="
" width="321" height="231" /><img src="
" width="321" height="244" />
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
<img src="
" width="781" height="496" />
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
<img src="
" width="256" height="80" /><img src="
" width="355" height="289" /><img src="
" width="295" height="266" />
PR + pBABE = pBABE/PR
Cut PR w/EcoRI
Cut pBABE w/EcoRI
Combine the insert and vector with T4 ligase
3.2 Gel electrophoresis
<img src="
" width="162" height="248" /><img src="
" width="164" height="247" hspace="100" />
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
<img src="
" width="558" height="349" />
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.
