1537 pT7A3-Klenow3'-5'exo–

Cloning of 1537 pT7A3-Klenow3'-5'exo–

Purpose: Cloning of 1537 pT7A3-Klenow3'-5'exo–

Date: 2013-2-4---2013-2-

Notebook:0

Page:

Clone ID:1537

Experimenters: Shanyi

Results

subcloning

subclone from 1530pET28b-Klenow(3'->5'_exo–) to 1279 pT7A3 using Nde1+ Sal1

	band 1	band 3
	1530 pET28b-Klenow(3'->5'_exo–)	1279pT7A3
enzymes	Nde1+ Sal1

Colony Screening

Date: 2013-2-

Make miniprep and digest with Nde1+ Sal1

Result intepretation/Summary

• <br>13-pT7A3-polyAPol

Protein Expression

2013/3/11

13-pT7A3-polyAPol

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, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.11 / 39375.95
3	pT7A3-Klenow	Klenow	1501	5 ml, Nico21(DE3)plysN	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, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.14 / 29824.85
5	pT7A3-DNA-Pol-1	DNA-Pol-1	1508	5 ml, Nico21(DE3)plysN	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
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

Protein Expression 2

2013/3/20
SHANYI

Lane	Name	Protein Name	Clone ID	Host	Induction	Purification	pI/Mw
M	PageRuler Plus Prestained protein ladder
1	pT7A3-hTdT	hTdT	1509	5 ml, Nico21(DE3)plysN	0.2 mM IPTG  RT o/n	Ni beads	8.66 / 58536.26
2	pET28b-T7-DNA-Polymerase	T7-DNA-Polymerase	1533	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	6.04 / 79691.65
3	pET28b-T7-DNA-Pol-D28	T7-DNA-Pol-D28	1539	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.90 / 76333.85
4	pT7A3-MBP-(3C)-Exo-III	MBB-(3C)-Exo-III	1569	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.80 / 30969.17+43.6
5	pT7A3-T4RNA-Lig2-truncated-KQ	T4RNA-Lig2-truncated-KQ	1581	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.14 / 29796.84
6	pT7A3-MBP-(3c)-Ecoli-UDG	MBP-(3c)-Ecoli-UDG	1591	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	6.67 / 25693.23 +43.6
7	pT7A3-MBP-(3c)-newPhi29	MBP-(3c)-newPhi29	1617	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	8.61 / 66714.05 +43.6
8	pT7A3-MBP-(3C)-Exo-1	MBP-(3C)-Exo-1	1570	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.51 / 54484.77 +43.6
9	pT7A3-Klenow	Klenow	1501	5 ml, Nico21(DE3)plysN	0.1 mM IPTG RT o/n	Ni beads	5.08 / 71328.35
10	pT7A3-Klenow(3'->5'_exo–)	Klenow(3'->5'_exo–)	1537	5 ml, Nico21(DE3)plysN	0.1 mM IPTG RT o/n	Ni beads	5.77 / 67969.77
11	pT7A3-Ecoli_RecA	Ecoli_RecA	1529	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.09 / 37973.37