1614-pCold-TF-(3c)
Cloning of 1614-pCold-TF-(3c)
[edit]Purpose: Cloning of 1614-pCold-TF-(3c)
Date: 2013-1-28---2013-1-31
Notebook:0
Page:
Clone ID:1614
Experimenters: yingying
Results
[edit]cloning
[edit]pcr1
make promoter by gene synthesis
pcr2
PCR TF from Trigger Factor chaperone plasmid
571 pG-Tf2
8His-TF-F+TF-3C-Nde1-R
pcr1+pcr2
Join two fragment using
Cold-1 + TF-3C-Nde1-R
Digest pcr product with BglII + Nde1.
Clone into BglII + Nde1 digested v19 pET28-cold
Colony Screening
[edit]Date: 2013-1
Make miniprep and digest with
Result intepretation/Summary
[edit]- <br>
Conclusion
[edit]<br>
<br>
<br>
<br>
Cloning of 1614-pCold-TF-(3c)
[edit]Purpose: Cloning of 1614-pCold-TF-(3c)
Date: 2013-2-1---2013-2-4
Notebook:0
Page:
Clone ID:1614
Experimenters: yingying
Results
[edit]cloning
[edit]pcr1
PCR from v19 pET28-cold using
Cold-BglII-F + 8His-TF-R
pcr2
PCR TF from Trigger Factor chaperone plasmid
571 pG-Tf2
8His-TF-F+TF-3C-Nde1-R
pcr1+pcr2
Join two fragment using
Cold-BglII-F + TF-3C-Nde1-R
Colony Screening
[edit]Date: 2013-2-4
Make miniprep and digest with BglII + Nde1
Result intepretation/Summary
[edit]- &lt;br&gt;
Conclusion
[edit]&lt;br&gt;
&lt;br&gt;
<br>
<br>
Cloning of 1614-pCold-TF-(3c)
[edit]Purpose: Cloning of 1614-pCold-TF-(3c)
Date: 2013-2-14------2013-2-25
Notebook:0
Page:
Clone ID:1614
Experimenters: yingying
Results
[edit]cloning
[edit]pcr1
PCR from v19 pET28-cold using
Cold-BglII-F + 8His-TF-R2
pcr2
PCR TF from Trigger Factor chaperone plasmid
571 pG-Tf2
8His-TF-F2+TF-3C-Nde1-R
pcr1+pcr2
Join two fragment using
Cold-BglII-F + TF-3C-Nde1-R
Colony Screening
[edit]Date: 2013-2-25
Make miniprep and digest with BglII + Nde1
Result intepretation/Summary
[edit]Conclusion
[edit]protein screening
[edit]
| lane |
name |
clone ID |
host |
induction |
purification |
MW | |
| 1 |
pT7A3-T5-Exo |
1567 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
21KD/5.95 | |
| 2 |
pCold-TF-(3c) |
1614 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
55.9KD/5.19 | |
| 3 |
pET28b-Nde1 |
1673 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
44KD/8.41 | |
| 4 |
pTCold-MBP-(3C) |
1677 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
44KD | |
| 5 |
pCold-C(3C)-MBP-6H |
1692 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
46KD/5.77 | |
| 6 |
pT7A3-wtSM2 |
1694 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads, should use CBD beads |
33KD/6.30 | |
| 7 |
pCold-MBP-(3C)-TEV |
1696 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
72KD/6.04 | |
| 8 |
pCold-GST-(3c)-wt3C |
1697 |
4 ml,NiCO21(DE3)pLysN |
0.2 mM IPTG 25 oC o/n |
Ni beads |
21KD/8.47 | |
| 9 |
pT7A3-MBP-(E3C)-p53wt |
1707 |
4 ml,
BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
88KD/5.76 | |
| 10 |
pET28b-hINFA2 |
1711 |
4 ml,
BL21(DE3)Star, pRare2-pLysS |
0.2 mM IPTG 25 oC o/n |
Ni beads |
21KD/5.95 | |