Crm at 15:15, 12 June 2017

2017-06-12T15:15:16Z

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== 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 
| -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 ==

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"
|-
| 
| 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

|-
|
ddH2O

|
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 -80oC.

== 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 
| -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 ==

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 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 ==

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

{| 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

|-
|
ddH2O

|
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 
| -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
[[Category:Reports]]

MS reports 6 5 2017-6 9 2017 - Revision history

Crm at 15:15, 12 June 2017