MS reports 5 23 2017-5 26 2017
05/23/2017
[edit]Plasmid transformation. 483/1464/1946/1497 transfer into Nico21(DE3) PlysN, culture with LB/K/C media.
Order shipment of 4865 (phi29 (φ29) DNA Polymerase).
Sorting refrigerator.
Order shipment of 4866 (HTN-GFP, Cell-Permeable Green Fluorescent Protein).
Culture 1613 1782 2196 1498 2295 for expression screening.
run SDS-PAGE gel 1811VPol / 1995Kfu/ 2409iFLP/ 2295yPolyA-Pol/ 1458KOD / 1715Pfu / 1315Mth RNA Ligase 05/23/2017
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
| sample |
1811 |
1995 |
2409 1 |
2409 2 |
2295 1 |
2295 2 |
1458 1 |
1458 2 |
MW |
1715 13 |
1715 16 |
1315 41 |
| uL |
0.2 |
0.4 |
1.8 |
1.8 |
1.5 |
1.2 |
0.2 |
0.4 |
10 |
0.8 |
1.3 |
0.5 |
| ddH2O |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
\ |
15 |
15 |
15 |
05/24/2017
[edit]Use IPTG to induce 1613pET28-cold-GST-3C 1782Tth-Ligase 2196PBCV-1-Lig 1498TaqDNALigase 2295yPolyA-Pol for expression screening. Spin down cells, wash with Lysis Buffer, break down through blunted needle, and incubate with 50 ul regenerated Ni beads. Wash Ni beads, prepare to run SDS-PAGE gel.
Select one colony to culture 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq in 15ml LB/K/C media.
Quote for Dr. Kato, GRred.
Deal with order 4867.
Data management.
Cell culture, 1708 HTNCre.
05/25/2017
[edit]Use IPTG to induce 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq for expression screening. Spin down cells, wash with Lysis Buffer, break down through blunted needle, and incubate with 50 ul regenerated Ni beads. Wash Ni beads, prepare to run SDS-PAGE gel.
Culture 1708HTNCre in 8L LB media. Induce with IPTG.
Culture ZXPmp / 818 / 819 / 820.
Dialysis of 1715Pfu and 1315 Mth RNA Ligase. The final concentration of 1715 Pfu is 4.10 mg/ml, totally 7ml, 28.70mg. The final concentration of 1315 Mth RNA Ligase is 6.50 mg/ml, totally 18.8ml, 122.20mg.
Small scale expression screening of 1613pET28-cold-GST-3C / 1782Tth-Ligase / 2196PBCV-1-Lig / 1498TaqDNALigase / 2295yPolyA-Pol. Run SDS-PAGE gel.
| number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
| sample |
1613 P |
1782 P |
2196 P |
1498 P |
2295 P |
MW |
1613 S |
1782 S |
2196 S |
1498 S |
2295 S |
| uL |
15 |
15 |
15 |
15 |
15 |
10 |
15 |
15 |
15 |
15 |
15 |
S: use lysis buffer S to break down cell, spin down and collect supernatant.
P: use lysis buffer P to wash sediment, sse blunted needle to break pellet, spin down and collect supernatant. 
Cell culture of 1782 Tth-Ligase
05/26/2017
[edit]Prepare LB media (20L).
Spin down 1708 HTNCre, wash with buffer A, break down through homogenizer, collect supernatant, load onto Ni column.
Troubleshoot with printer, load paper.
Deal with order 4868.
Small scale expression screening of 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq. Run SDS-PAGE gel.
| number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
| sample |
483 P |
1464 P |
1946 P |
1497 P |
MW |
483 S |
1464 S |
1946 S |
1497 S |
483 U |
1464 U |
1946 U |
| uL |
15 |
15 |
15 |
15 |
10 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
S: use lysis buffer S to break down cell, spin down and collect supernatant.
P: use lysis buffer P to wash sediment, sse blunted needle to break pellet, spin down and collect supernatant.
U: Uninduced sample. S and P induced with IPTG.
Lane 6~9 have bands. It means there is protein expression occur in both induced sample.
Lane 2 is brighter than lane 7, both of the two lanes load 1464 T7 DNA Ligase. It means there is more protein in the sediment than the supernatant.
There is no band in lane 10~12. Use these 3 lanes as negative control.