MS reports 6 26 2017-6 30 2017
06/26/2017
[edit]Culture 1458 KOD 200ml.
Culture 1518 T3 ligase 8L, induced with IPTG.
Homogenize 1697 GST-wt3C, load onto Ni column.
Load 1942 phi29 onto QHP column.
06/27/2017
[edit]Culture 1995 Kfu 200ml.
Culture 1458 KOD 8L, induced with IPTG.
Homogenize 1518 T3 ligase, load onto Ni column.
Elute 1697 GST-wt3C with Buffer B 06/27/2017
| -0.21 |
-0.21 |
-0.21 |
0.00 |
4.41 |
(1~5) |
| 3.66 |
1.16 |
0.60 |
0.35 |
0.24 |
(6~10) |
| 0.19 |
0.15 |
0.13 |
0.06 |
0.03 |
(11~15) |
collect 5~11.
Load 1697 GST-wt3C onto QHP column.
Elute 1942 phi29
06/28/2017
[edit]Culture 1811 VPol 200ml.
Culture 1995 Kfu 8L, induced with IPTG.
Homogenize 1458 KOD, load onto Ni column.
Elute 1518 T3 ligase with Buffer B 06/28/2017
| -0.22 |
-0.20 |
-0.18 |
0.09 |
1.22 |
(1~5) |
| 0.97 |
0.75 |
0.55 |
0.41 |
0.31 |
(6~10) |
| 0.22 |
0.15 |
0.11 |
0.05 |
0.03 |
(11~15) |
collect 5~11.
Load 1518 T3 ligase onto QHP column.
Elute 1697 GST-wt3C
Run SDS-PAGE gel of 1942 phi 29 5.72/70.6 06/28/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
|
sample |
11 |
12 |
13 |
14 |
15 |
MW |
|
uL |
12.5 |
4.0 |
4.2 |
5.3 |
8.4 |
10 |
|
ddH2O |
\ |
10 |
10 |
10 |
5 |
\ |
forward lane 1~2 to the next step.
Small scale screening of 2462 Tn5 transposase 8.41 / 83.8 818 N-6His 6.12 / 31.9 819 N-His 5.92 / 31.2 820 N-6His 5.99 / 31.2
Run SDS-PAGE gel 06/28/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
|
sample |
MW |
2462 |
818 |
819 |
820 |
|
uL |
10 |
15 |
15 |
15 |
15 |
2462 818 819 820 can do protein purification.
Run SDS-PAGE gel of 1697 GST-wt3C 8.48/21.4 06/28/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
18 |
19 |
20 |
22 |
23 |
MW |
24 |
25 |
26 |
27 |
29 |
31 |
|
uL |
1.3 |
0.9 |
0.9 |
0.8 |
0.8 |
10 |
1.0 |
1.4 |
1.8 |
2.4 |
4.6 |
7.7 |
|
ddH2O |
15 |
15 |
15 |
15 |
15 |
\ |
15 |
15 |
15 |
10 |
10 |
5 |
forward lane 1~2 to the next step.
06/29/2017
[edit]Culture 2462 Tn5 transposase 200ml.
Culture 1811 vPol 8L, induced with IPTG.
Homogenize 1995 Kfu, load onto Ni column.
Wash Ni column (load with 1458 KOD) with Ni washing Buffer/ Tris-HCl/ Buffer B, store at -80oC.
Elute 1458 KOD with Buffer B 06/29/2017
| -0.02 |
-0.00 |
-0.01 |
0.09 |
0.12 |
(1~5) |
| 4.98 |
18.72 |
18.77 |
11.26 |
6.98 |
(6~10) |
| 4.54 |
2.84 |
1.89 |
1.31 |
0.92 |
(11~15) |
| 0.72 |
0.58 |
0.51 |
0.44 |
0.39 |
(16~20) |
| 0.24 |
0.16 |
0.13 |
0.07 |
0.04 |
(21~25) |
collect 6~21.
Elute 1518 T3 ligase from QHP column.
Run SDS-PAGE gel of 1518 T3 ligase 5.62/41.6 06/29/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
26 |
28 |
29 |
30 |
31 |
32 |
33 |
34 |
MW |
35 |
36 |
37 |
|
uL |
6.7 |
4.8 |
4.2 |
3.4 |
3.5 |
2.5 |
2.0 |
1.8 |
10 |
1.9 |
2.6 |
3.7 |
|
ddH2O |
10 |
10 |
10 |
10 |
10 |
10 |
15 |
15 |
\ |
15 |
10 |
10 |
forward lane 10~12 to the next step.
06/30/2017
[edit]Spin down 1811 vPol and store at -20oC.
Wash Ni column (load with 1995 Kfu) with Ni washing Buffer/ Tris-HCl/ Buffer B, store at -80oC.
Elute 1995 Kfu with Buffer B 06/30/2017
| -0.21 |
-0.19 |
-0.09 |
0.02 |
1.26 |
(1~5) |
| 1.09 |
0.85 |
0.69 |
0.58 |
0.58 |
(6~10) |
| 0.58 |
0.56 |
0.55 |
0.54 |
0.51 |
(11~15) |
| 0.45 |
0.40 |
0.40 |
0.36 |
0.33 |
(16~20) |
| 0.32 |
0.30 |
0.21 |
0.14 |
0.08 |
(21~25) |
collect 5~22.
Prepare LB media 20L.
Dialysis of 1942 phi29, 1697 GST-wt3C, 1518 T3 ligase.
1942 phi29: 1.07mg/ml, 3.0ml, purity >90%.
1697 GST-wt3C: 3.03mg/ml, 4.0ml, purity >90%.
1518 T3 ligase: 2.50mg/ml, 3.5ml, purity >90%.