Editing MS reports 6 5 2017-6 9 2017

== 06/05/2017 ==

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

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.03<br/>
| -0.02<br/>
| -0.08<br/>
| -0.12<br/>
| -0.13<br/>
| (1~5)<br/>
|-
| -0.11<br/>
| -0.05<br/>
| 0.13<br/>
| 0.50<br/>
| 1.18<br/>
| (6~10)<br/>
|-
| 2.92<br/>
| 1.33<br/>
| 0.51<br/>
| 0.27<br/>
| 0.17<br/>
| (11~15)<br/>
|-
| 0.17<br/>
| 0.08<br/>
| 0.04<br/>
| 0.04<br/>
| 0.03<br/>
| (16~20)<br/>
|}

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 ==

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)

[[File:125-483 Tth Polymerase QHP 6 6 2017.jpg|RTENOTITLE]]



Run SDS-PAGE gel of 1782 Tth ligase and 483 Tth polymerase 06/06

{| style="width:500px" cellspacing="1" cellpadding="0" border="1" width="500"
|-
| <br/>
| colspan="3" | 
1782 Tth ligase 79.1

| 
| colspan="8" | 
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

|-
| 
ddH<sub>2</sub>O

| 
15

| 
15

| 
10

| 
\

| 
10

| 
15

| 
10

| 
10

| 
15

| 
10

| 
10

| 
10

|}



[[File:Protein Expression SDS-PAGE 124-1782 Tth Ligase and 125-483 Tth Polymerase QHP 6 6 2017.jpg|RTENOTITLE]]

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 -80<sup>o</sup>C.

== 06/07/2017 ==

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

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.19<br/>
| -0.21<br/>
| -0.02<br/>
| 1.36<br/>
| 2.34<br/>
| (1~5)<br/>
|-
| 7.36<br/>
| 3.66<br/>
| 1.11<br/>
| 0.65<br/>
| 0.34<br/>
| (6~10)<br/>
|-
| 0.15<br/>
| 0.09<br/>
| 0.06<br/>
| 0.03<br/>
| 0.01<br/>
| (11~15)<br/>
|}

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 ==

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)

[[File:126-1464 T7 DNA Ligase QHP 6 8 2017.jpg|RTENOTITLE]]

Elute SP FF column (load with 1708 Cre Recombinase) , combine fration with OD<sub>600</sub>>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 ==

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 -80<sup>o</sup>C.

Spin down 1498 Taq DNA Ligase, store at -20<sup>o</sup>C.

Run SDS-PAGE gel of 1464&nbsp;1464 T7 DNA Ligase 06/09

{| style="width:500px" cellspacing="1" cellpadding="0" border="1" width="500"
|-
| 
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

|-
| 
ddH<sub>2</sub>O

| 
5

| 
10

| 
15

| 
15

| 
15

| 
15

| 
15

| 
10

| 
\

|}

[[File:Protein Expression SDS-PAGE 126-1464 T7 DNA Ligase QHP 6 9 2017.jpg|RTENOTITLE]]

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

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.05<br/>
| -0.21<br/>
| -0.07<br/>
| 0.81<br/>
| 2.81<br/>
| (1~5)<br/>
|-
| 2.05<br/>
| 1.09<br/>
| 0.71<br/>
| 0.50<br/>
| 0.33<br/>
| (6~10)<br/>
|-
| 0.23<br/>
| 0.18<br/>
| 0.13<br/>
| 0.05<br/>
| 0.01<br/>
| (11~15)<br/>
|}

collect 4~12
[[Category:Reports]]