MS reports 5 15 2017-5 19 2017
05/15/2017
[edit]run SDS-PAGE gel 1518 T3 Ligase/1542 sso7d ToughTaq 05/15/2017
| 1518 T3 Ligase 41.6 |
1542 sso7d Native ToughTaq 72.0 | |||||||||||
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
| sample |
FT1 |
23 |
25 |
29 |
FT2 |
MW |
FT1 |
25 |
26 |
29 |
30 |
FT2 |
| uL |
5 |
2.8 |
1.2 |
0.4 |
15 |
10 |
5 |
0.6 |
0.5 |
0.5 |
0.4 |
15 |
| ddH2O |
10 |
10 |
15 |
15 |
\ |
\ |
10 |
15 |
15 |
15 |
15 |
\ |
Protein Expression Screening of 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
| sample |
483 1 |
1464 1 |
1946 1 |
1497 1 |
MW |
483 2 |
1464 2 |
1946 2 |
1497 2 |
1518 28 |
1518 30 |
1542 31 |
| uL |
15 |
15 |
15 |
15 |
10 |
15 |
15 |
15 |
15 |
1.3 |
0.8 |
0.8 |
| ddH2O |
\ |
\ |
\ |
\ |
\ |
\ |
\ |
\ |
\ |
15 |
15 |
15 |
1: induced with 0.5 mM IPTG
2: without induce
_1518(30)_1542(31)_05_15_2017.jpg)
Conclusion: 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq need to do from plasmid transformation. 1518(29, 30) and 1542( 29,30,31) can forward to the next step.
transfaction reagent 10ul/tube.
05/16/2017
[edit]transfaction reagent 100ul/tube.
wash Ni column with Buffer A/ Tris-HCl/Buffer B to elute 1715 Pfu.
1715 Pfu elute with Buffer B 05/16/2017
| 0.24 |
0.02 |
-0.20 |
-0.06 |
1.54 |
(1~5) |
| 4.14 |
3.55 |
2.67 |
2.13 |
1.66 |
(6~10) |
| 1.45 |
1.29 |
1.12 |
0.98 |
0.88 |
(11~15) |
| 0.71 |
0.63 |
0.56 |
0.53 |
0.46 |
(16~20) |
| 0.43 |
0.40 |
0.38 |
0.29 |
0.15 |
(21~25) |
Collect 5~23
Load 1715 Pfu onto QHP column.
Plasmid transformation. 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq transfer into NEB Turbo, culture with LB/K media.
Dialysis of 1518 T3 DNA Ligase and 1542 Native ToughTaq.
1518 total 6.5ml, 2.35mg/ml. 1542 total 7.7ml, 3.84mg/ml.
Load 1315 Mth RNA Ligase onto Ni Column.
Deal with order 4862.
05/17/2017
[edit]Culture 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq with 5ml LB/K media.
Wash Ni column with Buffer A/ Tris-HCl/Buffer B to elute 1315 Mth RNA Ligase.
1315 Mth RNA Ligase elute with Buffer B 05/17/2017
| 0.08 |
0.34 |
3.43 |
4.72 |
4.42 |
(1~5) |
| 3.33 |
2.43 |
1.68 |
1.32 |
1.07 |
(6~10) |
| 0.86 |
0.74 |
0.65 |
0.56 |
0.54 |
(11~15) |
| 0.47 |
0.46 |
0.26 |
0.18 |
0.11 |
(16~20) |
Collect 3~17.
Load 1315 Mth RNA Ligase onto QHP column.
Sorting warehouse.
prepare Buffer A /Buffer B/ Ni column regeneration Buffer.
purify 1715 Pfu through FPLC.
05/18/2017
[edit]plasmid extraction of 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq, and transfer into Nico21(DE3) PlynS.
purify 1315 Mth RNA Ligase through FPLC.
Sterilize 10ul tips.
05/19/2017
[edit]Prepare competent cell (Nico21)
Run SDS-PAGE gel of 121-1715 Pfu and 122-1315 Mth RNA Ligase.
| 1715 Pfu 98.6 |
1315 Mth RNA Ligase 45.3 | |||||||||||
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
| sample |
14 |
15 |
17 |
18 |
19 |
MW |
25 |
27 |
33 |
35 |
36 |
44 |
| uL |
0.4 |
1.0 |
0.9 |
0.4 |
0.8 |
10 |
0.9 |
1.0 |
0.5 |
0.2 |
0.2 |
0.9 |
| ddH2O |
15 |
15 |
15 |
15 |
15 |
\ |
15 |
15 |
15 | 15 |
15 |
15 |
1~5:1715
6:MW
7~12:1315
Conclusion: forward sample 14 and 15 of 1715, and 33~44 of 1315 to the next step.
Transfaction reagent 50ul/tube
QC of 1542 Native ToughTaq.
| 1 |
2 |
3 |
4 |
5 |
6 |
7 |
|
positive control |
1mg/ml |
0.1mg/ml |
10ug/ml |
1ug/ml |
0.1ug/ml |
negative control |
| 0.5ul |
0.5ul |
0.5ul |
0.5ul |
0.5ul |
0.5ul |
0 |
20ul/tube:
| dNTP |
template |
F |
R |
10x Buffer |
H2O |
Taq |
| 1ul |
0.1ul |
1ul |
1ul |
2ul |
14.4ul |
0.5ul |
Conclusion: need to do again with less cycle.