Editing MS reports 5 15 2017-5 19 2017

== 05/15/2017 ==

run SDS-PAGE gel '''<span style="color:#FF0000">1518 T3 Ligase/1542 sso7d ToughTaq</span>''' 05/15/2017

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| rowspan="1" colspan="5" | 1518 T3 Ligase 41.6<br/>
| <br/>
| rowspan="1" colspan="6" | 1542 sso7d Native ToughTaq 72.0<br/>
|-
| Number<br/>
| 1<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 5<br/>
| 6<br/>
| 7<br/>
| 8<br/>
| 9<br/>
| 10<br/>
| 11<br/>
| 12<br/>
|-
| sample<br/>
| FT1<br/>
| <span style="color:#000000">23</span><br/>
| 25<br/>
| 29<br/>
| FT2<br/>
| MW<br/>
| FT1<br/>
| 25<br/>
| 26<br/>
| 29<br/>
| 30<br/>
| FT2<br/>
|-
| uL<br/>
| 5<br/>
| 2.8<br/>
| 1.2<br/>
| 0.4<br/>
| 15<br/>
| 10<br/>
| 5<br/>
| 0.6<br/>
| 0.5<br/>
| 0.5<br/>
| 0.4<br/>
| 15<br/>
|-
| ddH<sub>2</sub>O<br/>
| 10<br/>
| 10<br/>
| 15<br/>
| 15<br/>
| \<br/>
| \<br/>
| 10<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| \<br/>
|}

[[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<br/>
| 1<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 5<br/>
| 6<br/>
| 7<br/>
| 8<br/>
| 9<br/>
| 10<br/>
| 11<br/>
| 12<br/>
|-
| sample<br/>
| 
483

1

| 
<span style="color:#000000">1464</span>

<span style="color:#000000">1</span>

| 
1946

1

| 
1497

1

| MW<br/>
| 
483

2

| 
1464

2

| 
1946

2

| 
1497

2

| 
1518

28

| 
1518

30

| 
1542

31

|-
| uL<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 10<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 1.3<br/>
| 0.8<br/>
| 0.8<br/>
|-
| ddH<sub>2</sub>O<br/>
| \<br/>
| \<br/>
| \<br/>
| \<br/>
| \<br/>
| \<br/>
| \<br/>
| \<br/>
| \<br/>
| 15<br/>
| 15<br/>
| 15<br/>
|}

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.

== <br/>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<br/>
| 0.02<br/>
| -0.20<br/>
| -0.06<br/>
| 1.54<br/>
| (1~5)<br/>
|-
| 4.14<br/>
| 3.55<br/>
| 2.67<br/>
| 2.13<br/>
| 1.66<br/>
| (6~10)<br/>
|-
| 1.45<br/>
| 1.29<br/>
| 1.12<br/>
| 0.98<br/>
| 0.88<br/>
| (11~15)<br/>
|-
| 0.71<br/>
| 0.63<br/>
| 0.56<br/>
| 0.53<br/>
| 0.46<br/>
| (16~20)<br/>
|-
| 0.43<br/>
| 0.40<br/>
| 0.38<br/>
| 0.29<br/>
| 0.15<br/>
| (21~25)<br/>
|}

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.

<span style="color:#FF0000">'''1518 total 6.5ml, 2.35mg/ml. 1542 total 7.7ml, 3.84mg/ml.'''</span>

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<br/>
| 0.34<br/>
| 3.43<br/>
| 4.72<br/>
| 4.42<br/>
| (1~5)<br/>
|-
| 3.33<br/>
| 2.43<br/>
| 1.68<br/>
| 1.32<br/>
| 1.07<br/>
| (6~10)<br/>
|-
| 0.86<br/>
| 0.74<br/>
| 0.65<br/>
| 0.56<br/>
| 0.54<br/>
| (11~15)<br/>
|-
| 0.47<br/>
| 0.46<br/>
| 0.26<br/>
| 0.18<br/>
| 0.11<br/>
| (16~20)<br/>
|}

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"
|-
| <br/>
| rowspan="1" colspan="5" | 1715 Pfu 98.6<br/>
| <br/>
| rowspan="1" colspan="6" | 1315 Mth RNA Ligase 45.3<br/>
|-
| Number<br/>
| 1<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 5<br/>
| 6<br/>
| 7<br/>
| 8<br/>
| 9<br/>
| 10<br/>
| 11<br/>
| 12<br/>
|-
| sample<br/>
| 14<br/>
| <span style="color:#000000">15</span><br/>
| 17<br/>
| 18<br/>
| 19<br/>
| MW<br/>
| 25<br/>
| 27<br/>
| 33<br/>
| 35<br/>
| 36<br/>
| 44<br/>
|-
| uL<br/>
| 0.4<br/>
| 1.0<br/>
| 0.9<br/>
| 0.4<br/>
| 0.8<br/>
| 10<br/>
| 0.9<br/>
| 1.0<br/>
| 0.5<br/>
| 0.2<br/>
| 0.2<br/>
| 0.9<br/>
|-
| ddH<sub>2</sub>O<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| 15<br/>
| \<br/>
| 15<br/>
| 15<br/>
| 15
| 15<br/>
| 15<br/>
| 15<br/>
|}

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<br/>
| 2<br/>
| 3<br/>
| 4<br/>
| 5<br/>
| 6<br/>
| 7<br/>
|-
| 
positive

control

| 1mg/ml<br/>
| 0.1mg/ml<br/>
| 10ug/ml<br/>
| 1ug/ml<br/>
| 0.1ug/ml<br/>
| 
negative

control

|-
| 0.5ul<br/>
| 0.5ul<br/>
| 0.5ul<br/>
| 0.5ul<br/>
| 0.5ul<br/>
| 0.5ul<br/>
| 0<br/>
|}

20ul/tube:

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

<br/>[[File:1542 Tough Taq QC 5 19 2017.JPG|RTENOTITLE]]

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