1123 pT7A3-MBP-(3C)-T4-RNA-Lig2

Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2

Purpose: Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2

Date: Jan 4, 2013

Notebook: 

Page:

Clone ID: 1123

Experimenters: Jennifer Truong

Results

Cloning

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

Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2

Purpose: Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2

Date: Jan 4, 2013

Notebook: 

Page:

Clone ID: 1123

Experimenters: Shanyi

Results

Cloning

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

send 1123-2 to sequeng, it is 1026. send 1123-4 to sequence again.

Protein Expression

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, Nico21(DE3)plysN	0.2 mM IPTG RT o/n	Ni beads	5.11 / 39375.95 +43.6
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 +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