1654 pT7A3-T3 DNA ligase

Cloning of 1654 pT7A3-T3 DNA ligase

Purpose: Cloning of 1654 pT7A3-T3 DNA ligase

Date: Jan , 2013

Notebook: 

Page:

Clone ID: 1654

Experimenters: Shanyi

Results

Cloning

PCR

Nde1 + Xho1

Colony screen

Cloning

PCR using contaminated T3 DNA ligase plasmid DNA as template and the following primers

New_T3_DNA_Lig-Nde1-F A1 cacgcaCATATGATGAACATCTTTAACACC

T3DNALigase-30 CTCGGAACTTCTCGAAGCTAGGGTGTCGCAGGGAGCCGTCCGGGGTCGCTTCCA
T3DNALigase-31 CTAGCTTCGAGAAGTTCCGAGGAACTGAGGACAACCCTCAAGAGAAAATGTAAC
T3DNALigase-32 TGATCGCTCGAGTTACATTTTCTCTTGAGGGTTGT

digest with Nde1 + Xho1

Colony screen

digest with Nde1 + Xho1

SEND 1654-3 TO SEQUENCE

Protein Expression

2013/3/29
SHANYI

Lane	Name	Protein Name	Clone ID	Host	Induction	Purification	pI/Mw
M	PageRuler Plus Prestained protein ladder
1	pT7A3-T4-G32	T4-G32	1097	5 ml, Nico21(DE3)plysN	0.2 mM IPTG  RT o/n	Ni beads	4.82 / 33506.43
2	pT7A3-MBP-(3c)-MutM	MBP-(3c)-MutM	1138	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	8.68 / 30290.07+43.6
3	pT7A3-KOD	KOD	1458	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	7.53 / 90022.42
4	pT7A3-UDG	UDG	1494	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.45 / 27174.73
5	pT7A3-T7-Endo1	T7-Endo1	1505	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	9.54 / 17171.84
6	pT7A3-polyAPol	polyAPol	1513	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	9.67 / 53870.91
7	pT7A3-T7-Exo	T7-Exo	1558	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	4.93 / 34502.20
8	pT7A3-MBP-(3C)-Exo-1	MBP-(3C)-Exo-1	1562	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	8.80 / 18143.72
9	pT7A3-MBP-(3C)-RnaseH	MBP-(3C)-RnaseH	1573	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	8.43 / 17596.99 +43.6
10	pT7A3-MBP-(3c)-E_coli_Endo IV	MBP-(3c)-E_coli_Endo IV	1593	5 ml, Nico21(DE3)plysN	0.2mM IPTG RT o/n	Ni beads	5.43 / 31479.53 +43.6
11	pT7A3-T3 DNA ligase	T3 DNA ligase	1654	5 ml, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.14 / 39350.70

Protein Purification

1. Step gradient on 10 ml HisTrap column

Buffer A: 10 mM imidazole, 20 mM Tris-HCl pH 8.0 , 500 mM NaCl, 0.05 mM EDTA, 1 mM beta-ME
Buffer B: 500 mM imidazole, 20 mM Tris-HCl pH 8, 300 mM NaCl, 0.05 mM EDTA, 1 mM beta-ME
method 64

SDS-PAGE: 2013/4/11

lane

1

2

3

4

5

6

7

8

10

11

12

clone ID

1458

Marker

1654

1697

name

pT7A3-KOD

pT7A3-T3 DNA ligase

pCold-GST-(3c)-wt3C

host

Nico21DE3plysN

Nico21DE3plysN

Nico21DE3plysN

induction

0.2 mM IPTG 25oC o/n

0.2 mM IPTG 25oC o/n

0.2 mM IPTG 25oC o/n

purification

10ml HisTrap HP

10ml HisTrap HP

10ml HisTrap HP

MW

7.53 / 90022.42

5.14 / 39350.70

21KD/8.47

Combine fraction 31-68, concentrate to 20ml

2. Gel filtration on TSK 250 gel filtration.

Buffer: 50 mM Na-Phosphate, pH 7.4, 200 mM NaCl

SDS-PAGE: 2013-4-17

lane	1		2	3	4	5	6		7	8	9	10	11		12
clone ID	1458						Marker		1654				1711		1712
name	pT7A3-KOD								pT7A3-T3 DNA  ligase				pET28b-hINFA2 h		pLambda-hINFA2
host	Nico21DE3plysN								Nico21DE3plysN
induction	0.2 mM IPTG 25oC o/n								0.2 mM IPTG 25oC o/n
purification	10ml HisTrap HP(2nd time)								Bio-Sil TSK 250
MW	7.53 / 90022.42								5.14 / 39350.70

concentrate fraction 28 ~31using spin concentator

3. Dialyze twice overnight.

Dialysis Buffer:20 mM Tris-HCl pH7.5, 50 mM NaCl, 50% Glycerol , 0.1 mM EDTA

4.  Store in a buffer (20 mM Tris-HCl pH7.5, 50 mM NaCl, 50% Glycerol, 0.1 mM EDTA, 1mM DTT) at -80oC (T7ligase box).

Concentration : 2 mg/ml