MS reports 6 5 2017-6 9 2017
06/05/2017
[edit]Wash Ni column (load with 483 Tth Polymerase) with Ni washing buffer / Tris-HCl / Buffer B, dilute and load onto QHP column.
Elute 483 Tth Polymerase with Buffer B 06/05/2017
| -0.03 |
-0.02 |
-0.08 |
-0.12 |
-0.13 |
(1~5) |
| -0.11 |
-0.05 |
0.13 |
0.50 |
1.18 |
(6~10) |
| 2.92 |
1.33 |
0.51 |
0.27 |
0.17 |
(11~15) |
| 0.17 |
0.08 |
0.04 |
0.04 |
0.03 |
(16~20) |
Culture 1464 T7 DNA Ligase 8L, induce with IPTG.
Culture 1946 TdT bovine 200ml.
Prepare LB/Amp agar.
Plasmid transformation of ligation sample 1 and 2.
Mini prep of 1420
Prepare LB media.
Deal with order 4873.
06/06/2017
[edit]Spin down 1464 T7 DNA Ligase, homogenize, centrifugate and load onto Ni column.
Culture 1946 TdT bovine 8L, induce with IPTG.
Culture 1497 sso7d-ToughTaq 200ml.
Elute QHP column (load with 483 Tth Polymerase)
Run SDS-PAGE gel of 1782 Tth ligase and 483 Tth polymerase 06/06
|
1782 Tth ligase 79.1 |
483 Tth polymerase 96.1 | |||||||||||
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
sample |
11 |
12 |
16 |
MW |
10 |
12 |
14 |
15 |
16 |
18 |
19 |
20 |
|
uL |
0.6 |
0.2 |
2.7 |
10 |
2.4 |
1.6 |
2.0 |
2.2 |
1.6 |
2.1 |
3.2 |
6.7 |
|
ddH2O |
15 |
15 |
10 |
\ |
10 |
15 |
10 |
10 |
15 |
10 |
10 |
10 |
Dialyse sample 11 and 12 of 1782 Tth ligase, 19 and 20 of 483 Tth polymerase.
1782 Tth ligase totally have 10ml, 10.64mg/ml, 106.4mg. 483 Tth polymerase totally have 13ml, 1.11mg/ml. 14.43mg. Store at -80oC.
06/07/2017
[edit]Culture 1498 Taq DNA Ligase 200ml.
Culture 1497 sso7d-ToughTaq 8L, induce with IPTG.
Spin down 1946 TdT bovine.
Prepare LB media 20L.
Prepare LB/K agar and LB/K/C agar.
Plasmid transformation. 1942 phi 29, 1601 6His-GST-i3C, 1518 T3 Ligase, 1765 T4 DNA Polymerase.
Wash Ni column (load with 1464 T7 DNA Ligase) with Ni washing buffer / Tris-HCl / Buffer B, dilute and load onto QHP column.
Elute 1464 T7 DNA Ligase with Buffer B 06/07/2017
| -0.19 |
-0.21 |
-0.02 |
1.36 |
2.34 |
(1~5) |
| 7.36 |
3.66 |
1.11 |
0.65 |
0.34 |
(6~10) |
| 0.15 |
0.09 |
0.06 |
0.03 |
0.01 |
(11~15) |
Collect 4~11.
1708 Cre Recombinase 1:1 dilute with SP Dilution Buffer. SP FF column wash with SP Elution Buffer and SP Wash Buffer, then load Cre onto SP FF column.
06/08/2017
[edit]Culture 1483 T4 DNA Ligase 200ml.
Culture 1498 Taq DNA Ligase 8L, induce with IPTG.
Spin down 1497 sso7d-ToughTaq.
1946 TdT bovine homogenize, centrifugate and load onto Ni column.
1942 phi 29, 1601 6His-GST-i3C, 1518 T3 Ligase, 1765 T4 DNA Polymerase pick up single colony each to culture in LB/K media for glycerol stock.
1942 phi 29, 1601 6His-GST-i3C, 1518 T3 Ligase, 1765 T4 DNA Polymerase, transfer into Nico21, culture on LB/K/C agar over night.
Elute QHP column (load with 1464 T7 DNA Ligase)
Elute SP FF column (load with 1708 Cre Recombinase) , combine fration with OD600>2.5, dialysis through dialysis bag against Cre dialysis buffer.
Prepare Storage buffer, SP Wash Buffer, SP Elution Buffer.
Lable transfection reagent.
06/09/2017
[edit]1518 T3 Ligase, 1601 6His-GST-i3C, 1765 T4 DNA Polymerase, 1942 phi 29 pick up single colony to culture in LB/K/C media for small scale screening.
Spin down 1518 T3 Ligase, 1601 6His-GST-i3C, 1765 T4 DNA Polymerase, 1942 phi 29 to make glycerol stock. Store at -80oC.
Spin down 1498 Taq DNA Ligase, store at -20oC.
Run SDS-PAGE gel of 1464 1464 T7 DNA Ligase 06/09
|
Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
|
sample |
13 |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
MW |
|
uL |
9.1 |
3.1 |
0.8 |
0.4 |
0.3 |
0.9 |
1.5 |
3.7 |
10 |
|
ddH2O |
5 |
10 |
15 |
15 |
15 |
15 |
15 |
10 |
\ |
Collect 4~8 forward to the next step. Dialysis.
23.2mg/ml, 4.5ml, 104.4mg. Purity>95%.
Wash Ni column (load with 1946 TdT bovine) with Ni washing buffer / Tris-HCl / Buffer B.
Elute 1946 TdT bovine with Buffer B 06/09/2017
| -0.05 |
-0.21 |
-0.07 |
0.81 |
2.81 |
(1~5) |
| 2.05 |
1.09 |
0.71 |
0.50 |
0.33 |
(6~10) |
| 0.23 |
0.18 |
0.13 |
0.05 |
0.01 |
(11~15) |
collect 4~12