Crm at 21:02, 24 May 2017

2017-05-24T21:02:09Z

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== 05/15/2017 ==

run SDS-PAGE gel '''1518 T3 Ligase/1542 sso7d ToughTaq''' 05/15/2017

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 
| rowspan="1" colspan="5" | 1518 T3 Ligase 41.6 
| 
| rowspan="1" colspan="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 
| \ 
|}

[[File:Protein Expression SDS-PAGE 119-1518 T3 DNA Ligase and 120-1542 Native ToughTaq QHP 5 15 2017.jpg|RTENOTITLE]]

Protein Expression Screening of 483 Tth Polymerase /1464 T7 DNA Ligase / 1946 TdT bovine / 1497 ToughTaq

{| cellspacing="1" cellpadding="1" border="1"
|-
| 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

[[File:Protein Screening 483 1464 1946 1497 1518(28) 1518(30) 1542(31) 05 15 2017.jpg|RTENOTITLE]]

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 ==

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

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

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

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

[[File:121-1715 Pfu QHP 5 17 2017.jpg|RTENOTITLE]]

== ==

== ==

== 05/18/2017 ==

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.

[[File:122-1315 Mth RNA Ligase QHP 5 18 2017.jpg|RTENOTITLE]]

Sterilize 10ul tips.

== ==

== ==

== 05/19/2017 ==

Prepare competent cell (Nico21)

Run SDS-PAGE gel of 121-1715 Pfu and 122-1315 Mth RNA Ligase.

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 
| rowspan="1" colspan="5" | 1715 Pfu 98.6 
| 
| rowspan="1" colspan="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

[[File:Protein Expression SDS-PAGE 121-1715 Pfu and 122-1315 Mth RNA Ligase QHP 5 19 2017.jpg|RTENOTITLE]]

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.

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

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| dNTP 
| template 
| F 
| R 
| 10x Buffer 
| H2O 
| Taq 
|-
| 1ul 
| 0.1ul 
| 1ul 
| 1ul 
| 2ul 
| 14.4ul 
| 0.5ul 
|}

 [[File:1542 Tough Taq QC 5 19 2017.JPG|RTENOTITLE]]

Conclusion: need to do again with less cycle.
[[Category:Reports]]

MS reports 5 15 2017-5 19 2017 - Revision history

Crm at 21:02, 24 May 2017