MS reports 6 12 2017-6 16 2017
06/12/2017
[edit]Culture 2295 yPolyA 200ml.
Culture 1483 T4 DNA Ligase 8L, induced with IPTG.
Homogenize 1497 ToughTaq, load onto Ni column.
Dilute 1946 TdT bovine and load onto QHP column.
Induce 1518 T3 Ligase 1601 6His-GST-i3C 1765 T4 DNA Polymerase 1942 phi29 with IPTG for small scale screening.
Prepare LB media 20L.
06/13/2017
[edit]Culture 2295 yPolyA 8L, induced with IPTG.
Spin down 1483 T4 DNA Ligase.
Wash Ni column (bind with 1497 ToughTaq) with Ni washing buffer, Tris-HCl, Buffer B, dilute and load onto QHP column.
Elute 1497 ToughTaq with Buffer B 06/13/2017
| -0.38 |
-0.09 |
0.05 |
0.91 |
1.59 |
(1~5) |
| 1.20 |
0.61 |
0.36 |
0.26 |
0.18 |
(6~10) |
| 0.15 |
0.11 |
0.08 |
0.06 |
0.05 |
(11~15) |
collect 4~11.
Elute QHP column (load with 1946 TdT bovine)
Resuspend 1498 Taq DNA Ligase, homogenize, load onto Ni column.
Spin down 1518 T3 Ligase 1601 6His-GST-i3C 1765 T4 DNA Polymerase 1942 phi29, incubate with Ni beads.
06/14/2017
[edit]Spin down 2295 yPolyA.
Resuspend 1483 T4 DNA Ligase, homogenize, load onto Ni column.
Wash Ni column (bind with 1498 Taq DNA Ligase) with Ni washing buffer, Tris-HCl, Buffer B, dilute and load onto QHP column.
Elute 1498 Taq DNA Ligase with Buffer B 06/14/2017
| 0.04 |
0.08 |
0.95 |
1.25 |
1.98 |
(1~5) |
| 2.84 | 4.72 |
2.16 |
1.15 |
0.78 |
(6~10) |
| 0.57 |
0.41 |
0.20 |
0.16 |
0.05 |
(11~15) |
collect 3~12.
Elute QHP column (load with 1497 ToughTaq)
Resuspend 1483 T4 DNA Ligase, homogenize, load onto Ni column.
Run SDS-PAGE gel of 1946 TdT bivine 7.67/45.7 06/14/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
18 |
19 |
21 |
23 |
MW |
24 |
26 |
27 |
28 |
31 |
33 |
34 |
|
uL |
5.6 |
4.4 |
1.0 |
1.7 |
10 |
1.3 |
0.7 |
0.9 |
0.9 |
2.5 |
4.8 |
6.7 |
|
ddH2O |
10 |
10 |
15 |
15 |
\ |
15 |
16 |
15 |
15 |
10 |
10 |
10 |
Forward sample 18~21 to the next step.
Run SDS-PAGE gel of 1518 T3 Ligase 5.62/41.6 1601 6His-GST-i3C 6.41/50.8 1765 T4 DNA Polymerase 6.19/105.9 1942 phi29 5.72/70.6 06/14/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
1518 S |
1601 S |
1765 S |
1942 S |
MW |
1518 P |
1601 P |
1765 P |
1942 P |
1518 U |
1601 U |
1765 U |
|
uL |
15 |
15 |
15 |
15 |
10 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
S:supernatant
P:sediment
U:uninduced
Only 1518 T3 Ligase and 1942 phi29 can do protein expression.
06/15/2017
[edit]Troubleshoot the low flow speed problem of Ni column. Wash Ni column (bind with 1483 T4 DNA Ligase) with Ni washing buffer.
Elute QHP column (load with 1498 Taq DNA Ligase)
Run SDS-PAGE gel of 1497 ToughTaq 7.40/72.2 06/15/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
22 |
23 |
25 |
MW |
26 |
27 |
28 |
29 |
30 |
31 |
33 |
35 |
|
uL |
3.5 |
0.7 |
2.7 |
10 |
2.2 |
3.0 |
2.2 |
2.4 |
2.2 |
1.6 |
2.2 |
4.6 |
|
ddH2O |
10 |
15 |
10 |
\ |
10 |
10 |
10 |
10 |
10 |
15 |
10 |
10 |
Forward sample 22~24 to the next step.
Prepare Buffer A.
Run SDS-PAGE gel of 1498 Taq DNA Ligase 6.56 / 79.1 06/15/2017
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
20 |
22 |
23 |
24 |
26 |
27 |
MW |
28 |
29 |
32 |
35 |
39 |
|
uL |
4.8 |
0.8 |
0.3 |
0.2 |
0.6 |
0.9 |
10 |
0.8 |
0.7 |
1.4 |
2.7 |
4.2 |
|
ddH2O |
10 |
15 |
15 |
15 |
15 |
15 |
\ |
15 |
15 |
15 |
10 |
10 |
Forward sample 37~39 to the next step.
Dialysis. 1946 TdT bovine, 3.3mg/ml, 2.0ml, 6.6mg. Store at -80oC, 1-1-2 (Enzyme 2017)
06/16/2017
[edit]Dialysis. 1497 ToughTaq, 4.5mg/ml, 2.7ml. 95%. 1498 Taq DNA ligase, 1.5mg/ml, 3.3ml. 90%. Store at -80oC, 1-1-2 (Enzyme 2017)
Sterilize tips.
Prepare QHP Washing Buffer and QHP Elution Buffer.
Data management.