1123 pT7A3-MBP-(3C)-T4-RNA-Lig2
Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2
[edit]Purpose: Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2
Date: Jan 4, 2013
Notebook:
Page:
Clone ID: 1123
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]Date: Jan 4, 2013
PCR using 1uL of 1:10 dilution of T4 genomic DNA and the following primers
T4RNA-lig-Nde1-F
atcgac CATatg tttaaaaagtatagcagtc
T4RNA-lig-Xho1-R
(cttggattgagttggtaagttaaCTCGAG atcgaa)
ttcgatCTCGAGttaacttaccaactcaatccaag
See lane 1
Cut out band, and purify
Digest insert (lane ) and vector (1514 pT7A3-MBP-(3C) lane ) with RE Nde1 and Xho1
Cut out bands, ligate together and transform
Screening
[edit]Result intepretation/Summary
[edit]Conclusion
[edit]
Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2
[edit]Purpose: Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2
Date: Jan 4, 2013
Notebook:
Page:
Clone ID: 1123
Experimenters: Shanyi
Results
[edit]Cloning
[edit]PCR
Cut out band, and purify
Digest insert (lane ) and vector (1514 pT7A3-MBP-(3C) lane ) with RE Nde1 and Xho1
4.92 / 24309.87
Cut out bands, ligate together and transform
PCR
Screening
[edit]
send 1123-2 to sequeng, it is 1026. send 1123-4 to sequence again.
Result intepretation/Summary
[edit]Conclusion
[edit]
Protein Expression
[edit]2013/3/11
| Lane |
Name |
Protein Name |
Clone ID |
Host |
Induction |
Purification |
pI/Mw |
| M |
Invitrogen SeeBlue plus2 |
|
|||||
| 1 |
pT7A3-T4_RNA_Ligase_1 |
T4_RNA_Ligase_1 |
1049 |
5 ml, Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
4.63 / 47485.21 |
| 2 |
pT7A3-MBP-(3C)-T4-RNA-Lig2 |
T4-RNA-Lig2 |
1123 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
5.11 / 39375.95 +43.6 |
| 3 |
pT7A3-Klenow |
Klenow |
1501 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
5.11 / 71294.91 |
| 4 |
pT7A3-T4_RNA_Ligase_ 2_Truncated |
T4_RNA_Ligase_ 2_Truncated |
1503 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
5.14 / 29824.85 |
| 5 |
pT7A3-DNA-Pol-1 |
DNA-Pol-1 |
1508 |
5 ml, |
0.2 mM IPTG RT o/n |
Ni beads |
5.40 / 103118.12 |
| 6 |
pT7A3-Phi29DNAPol |
Phi29DNAPol |
1512 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
8.61 / 66714.05 |
| 7 |
pT7A3-polyAPol |
polyAPol |
1513 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
9.67 / 53870.91 |
| 8 |
pT7A3-Klenow(3'->5'_exo–) |
Klenow(3'->5'_exo–) |
1537 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
5.77 / 67969.77 |
| 9 |
pT7A3-MBP-(3C)-Lambda-Exo |
Lambda-Exo |
1557 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
5.36 / 25925.62 +43.6 |
| 10 |
pT7A3-MBP-(3c)-Endonuclease VIII |
Endonuclease VIII |
1572 |
5 ml,
Nico21(DE3)plysN |
0.2 mM IPTG RT o/n |
Ni beads |
7.77 / 29845.13+43.6 |