1030-pHis-GST-TEV

Cloning of pHis-GST-TEV (1030)
Purpose: to clone pHis-GST-TEV
Date: 2012-3-26 to 2012-4-11
Notebook: Xiaojing Wang
Page: 004206
Clone ID: 1030
Experimenters: Xiaojing Wang
Results

The bands show PCR product of 1030,

TEV-Nde1-F
atcatg CATATG GGAGAAAGCTTGTTTAAGGG

TEV-Xho1-R
(cagttaaggaagcgactcaatag ctcgag actgac)

gtcagtctcgagctattgagtcgcttccttaactg

Digest result:

digest PCR product and vector with Nde1+Xho1, run gel

the left bands show 1030 insert, the right bands show 1029 vector

gel purify, ligate, transform Top10,K+

Colony Screening

1032-1,-2,-3,-4 may be correct.

Select 1032-4 to sequence.

Wrong design
should not use TEV-Nde1-F

Results

The bands show PCR product of 1030,

Use primer:
TEV-EcoR1-F
TATCG GAA TTC GGA GAA AGC TTG TTT AAG G
E F G E S L F K
TEV-Xho1-R
Xho1
(cagttaaggaagcgactcaatag ctcgag actgac)

gtcagtctcgagctattgagtcgcttccttaactg

Digest result:

digest PCR product and vector with EcoR1+Xho1, run gel

the left bands show 1030 insert, the right bands show 1029 vector

gel purify, ligate, transform Top10,K+

Colony Screening

Select 10 clones to do PCR Screening

Clone 2,3,4,5,6,7,8,10 may be correct.

Select Clone 2,3,4,5 to mini-prep.

Miniprep digestion 4/7/2012

Digest 1030 plasmid with Nco1 + EcoR1 + Xho1, why there is only 1 insert band?

4/9/2012

digest 1030 miniprep , #2 and #3 (above) with Xho1, EcoR, Nco1

Lane #	1	2	3	4	5	6	7
Sample #	3	3	3	4	4	4	pET28b vector
Digestion	Xho1-EcoR1	Nco1-Xho1	Nco1-EcoR1	Xho1-EcoR1	Nco1-Xho1	Nco1-EcoR1	Nco1-Xho1
Insert	TEV	GST-TEV	GST	TEV	GST-TEV	GST	x

sequencing: #3 with T7 term: good

4/17/2012 Expression in Nico21(DE3) cells:

1, 2, 5, 6, 7, 8, 9: after Ni and Glutathione beads

3, 4, 10,11, 12, 13,14, 15: After Ni beads