Crm at 20:28, 4 July 2017

2017-07-04T20:28:18Z

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== 06/26/2017 ==

Culture 1458 KOD 200ml.

Culture 1518 T3 ligase 8L, induced with IPTG.

Homogenize 1697 GST-wt3C, load onto Ni column.

Load 1942 phi29 onto QHP column.

== 06/27/2017 ==

Culture 1995 Kfu 200ml. Culture 1458 KOD 8L, induced with IPTG.

Homogenize 1518 T3 ligase, load onto Ni column.

Elute 1697 GST-wt3C with Buffer B 06/27/2017

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.21 
| -0.21 
| -0.21 
| 0.00 
| 4.41 
| (1~5) 
|-
| 3.66 
| 1.16 
| 0.60 
| 0.35 
| 0.24 
| (6~10) 
|-
| 0.19 
| 0.15 
| 0.13 
| 0.06 
| 0.03 
| (11~15) 
|}

collect 5~11.

Load 1697 GST-wt3C onto QHP column.

Elute 1942 phi29

[[File:133-1942 phi29 QHP 6 27 2017.jpg|RTENOTITLE]]

== 06/28/2017 ==

Culture 1811 VPol 200ml. Culture 1995 Kfu 8L, induced with IPTG.

Homogenize 1458 KOD, load onto Ni column.

Elute 1518 T3 ligase with Buffer B 06/28/2017

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.22 
| -0.20 
| -0.18 
| 0.09 
| 1.22 
| (1~5) 
|-
| 0.97 
| 0.75 
| 0.55 
| 0.41 
| 0.31 
| (6~10) 
|-
| 0.22 
| 0.15 
| 0.11 
| 0.05 
| 0.03 
| (11~15) 
|}

collect 5~11.

Load 1518 T3 ligase onto QHP column.

Elute 1697 GST-wt3C

[[File:134-1697 GST-wt3C QHP 6 28 2017.jpg|RTENOTITLE]]

Run SDS-PAGE gel of 1942 phi 29 5.72/70.6   06/28/2017

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

|
1

|
2

|
3

|
4

|
5

|
6

|-
|
sample

|
11

|
12

|
13

|
14

|
15

|
MW

|-
|
uL

|
12.5

|
4.0

|
4.2

|
5.3

|
8.4

|
10

|-
|
ddH2O

|
\

|
10

|
10

|
10

|
5

|
\

|}

[[File:Protein Expression SDS-PAGE 133-1942 phi29 QHP 6 27 2017.jpg|RTENOTITLE]]

forward lane 1~2 to the next step.

Small scale screening of 2462 Tn5 transposase 8.41 / 83.8    818 N-6His 6.12 / 31.9    819 N-His 5.92 / 31.2    820 N-6His 5.99 / 31.2

Run SDS-PAGE gel   06/28/2017

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

|
1

|
2

|
3

|
4

|
5

|-
|
sample

|
MW

|
2462

|
818

|
819

|
820

|-
|
uL

|
10

|
15

|
15

|
15

|
15

|}

[[File:Protein Screening 2462 818 819 820 06 28 2017.jpg|RTENOTITLE]]

2462 818 819 820 can do protein purification.

Run SDS-PAGE gel of 1697 GST-wt3C 8.48/21.4   06/28/2017

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

|
1

|
2

|
3

|
4

|
5

|
6

| 7 
| 8 
| 9 
| 10 
| 11 
| 12 
|-
|
sample

|
18

|
19

|
20

|
22

|
23

|
MW

| 24 
| 25 
| 26 
| 27 
| 29 
| 31 
|-
|
uL

|
1.3

|
0.9

|
0.9

|
0.8

|
0.8

|
10

| 1.0 
| 1.4 
| 1.8 
| 2.4 
| 4.6 
| 7.7 
|-
|
ddH2O

|
15

|
15

|
15

|
15

|
15

|
\

| 15 
| 15 
| 15 
| 10 
| 10 
| 5 
|}

[[File:Protein Expression SDS-PAGE 134-1697 GST-wt3C QHP 6 28 2017.jpg|RTENOTITLE]]

forward lane 1~2 to the next step.

== 06/29/2017 ==

Culture 2462 Tn5 transposase 200ml. Culture 1811 vPol 8L, induced with IPTG.

Homogenize 1995 Kfu, load onto Ni column.

Wash Ni column (load with 1458 KOD) with Ni washing Buffer/ Tris-HCl/ Buffer B, store at -80oC.

Elute 1458 KOD with Buffer B 06/29/2017

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.02 
| -0.00 
| -0.01 
| 0.09 
| 0.12 
| (1~5) 
|-
| 4.98 
| 18.72 
| 18.77 
| 11.26 
| 6.98 
| (6~10) 
|-
| 4.54 
| 2.84 
| 1.89 
| 1.31 
| 0.92 
| (11~15) 
|-
| 0.72 
| 0.58 
| 0.51 
| 0.44 
| 0.39 
| (16~20) 
|-
| 0.24 
| 0.16 
| 0.13 
| 0.07 
| 0.04 
| (21~25) 
|}

collect 6~21.

Elute 1518 T3 ligase from QHP column.

[[File:135-1518 T3 ligase QHP 6 29 2017.jpg|RTENOTITLE]]

Run SDS-PAGE gel of 1518 T3 ligase 5.62/41.6   06/29/2017

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

|
1

|
2

|
3

|
4

|
5

|
6

|
7

|
8

|
9

|
10

|
11

|
12

|-
|
sample

|
26

|
28

|
29

|
30

|
31

|
32

|
33

|
34

|
MW

|
35

|
36

|
37

|-
|
uL

|
6.7

|
4.8

|
4.2

|
3.4

|
3.5

|
2.5

|
2.0

|
1.8

|
10

|
1.9

|
2.6

|
3.7

|-
|
ddH2O

|
10

|
10

|
10

|
10

|
10

|
10

|
15

|
15

|
\

|
15

|
10

|
10

|}

 [[File:Protein Expression SDS-PAGE 135-1518 T3 ligase QHP 6 29 2017.jpg|RTENOTITLE]]

forward lane 10~12 to the next step.

== 06/30/2017 ==

Spin down 1811 vPol and store at -20oC.

Wash Ni column (load with 1995 Kfu) with Ni washing Buffer/ Tris-HCl/ Buffer B, store at -80oC.

Elute 1995 Kfu with Buffer B 06/30/2017

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| -0.21 
| -0.19 
| -0.09 
| 0.02 
| 1.26 
| (1~5) 
|-
| 1.09 
| 0.85 
| 0.69 
| 0.58 
| 0.58 
| (6~10) 
|-
| 0.58 
| 0.56 
| 0.55 
| 0.54 
| 0.51 
| (11~15) 
|-
| 0.45 
| 0.40 
| 0.40 
| 0.36 
| 0.33 
| (16~20) 
|-
| 0.32 
| 0.30 
| 0.21 
| 0.14 
| 0.08 
| (21~25) 
|}

collect 5~22.

Prepare LB media 20L.

Dialysis of 1942 phi29, 1697 GST-wt3C, 1518 T3 ligase.

1942 phi29: 1.07mg/ml, 3.0ml, purity >90%.

1697 GST-wt3C: 3.03mg/ml, 4.0ml, purity >90%.

1518 T3 ligase: 2.50mg/ml, 3.5ml, purity >90%.
[[Category:Reports]]

MS reports 6 26 2017-6 30 2017 - Revision history

Crm at 20:28, 4 July 2017