Paul.xu at 19:47, 22 April 2013

2013-04-22T19:47:40Z

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== '''Cloning of 1654 pT7A3-T3 DNA ligase''' ==

'''Purpose:''' Cloning of 1654 pT7A3-T3 DNA ligase

'''Date: '''Jan , 2013 

'''Notebook: '''

'''Page:'''

'''Clone ID: 1654'''

'''Experimenters: '''Shanyi

== '''Results''' ==

=== contamination by accident(1532-4) ===

=== Cloning ===

PCR 

[[Image:1532 1538-2 1560 1561-1562 1563pcr.JPG]] 

Nde1 + Xho1 

=== Colony screen ===

[[Image:1529 1532 1538digest.JPG]]

=== Cloning ===

PCR using contaminated T3 DNA ligase plasmid DNA as template and the following primers

New_T3_DNA_Lig-Nde1-F A1 cacgcaCATATGATGAACATCTTTAACACC

T3DNALigase-30 CTCGGAACTTCTCGAAGCTAGGGTGTCGCAGGGAGCCGTCCGGGGTCGCTTCCA T3DNALigase-31 CTAGCTTCGAGAAGTTCCGAGGAACTGAGGACAACCCTCAAGAGAAAATGTAAC T3DNALigase-32 TGATCGCTCGAGTTACATTTTCTCTTGAGGGTTGT 

[[Image:1654pcr.JPG]] 

digest with Nde1 + Xho1 

[[Image:Digest for1285 1535 1654.JPG]]

 

=== Colony screen ===

digest with Nde1 + Xho1 

[[Image:1654digest.JPG]][[Image:1654-1 -2 -6DIGEST.JPG]] 

SEND 1654-3 TO SEQUENCE

== Protein Expression ==

2013/3/29 SHANYI

[[Image:1097 1138 1458 1494 1505 1513 1558 1562 1573 1593 1654NEW.jpg]] 

{| width="832" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Name 
| Protein Name 
| Clone ID 
| Host 
| Induction 
| Purification 
| pI/Mw 
|-
| M 
| PageRuler Plus Prestained protein ladder 
|                            
| 
| 
| 
| 
| 
|-
| 1 
| pT7A3-T4-G32 
| T4-G32 
| 1097 
|
5 ml, 

Nico21(DE3)plysN

| 0.2 mM IPTG  RT o/n 
| Ni beads 
|
4.82 / 33506.43

|-
| 2 
| pT7A3-MBP-(3c)-MutM 
| MBP-(3c)-MutM 
| 1138 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
8.68 / 30290.07+43.6 

|-
| 3 
| pT7A3-KOD 
| KOD 
| 1458 
|
  5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
7.53 / 90022.42

|-
| 4 
| pT7A3-UDG 
| UDG 
| 1494 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
5.45 / 27174.73

|-
| 5 
| pT7A3-T7-Endo1 
| T7-Endo1 
| 1505 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
9.54 / 17171.84   

|-
| 6 
| pT7A3-polyAPol 
| polyAPol 
| 1513 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
9.67 / 53870.91 

|-
| 7 
| pT7A3-T7-Exo 
| T7-Exo 
| 1558 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
4.93 / 34502.20

|-
| 8 
| pT7A3-MBP-(3C)-Exo-1 
| MBP-(3C)-Exo-1 
| 1562 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
8.80 / 18143.72 

|-
| 9 
| pT7A3-MBP-(3C)-RnaseH 
| MBP-(3C)-RnaseH 
| 1573 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
8.43 / 

17596.99 +43.6

|-
| 10 
| pT7A3-MBP-(3c)-E_coli_Endo IV 
| MBP-(3c)-E_coli_Endo IV 
| 1593 
|   5 ml,
Nico21(DE3)plysN 

| 0.2mM IPTG RT o/n 
| Ni beads 
|
5.43 / 

31479.53 +43.6

|-
| 11 
| pT7A3-T3 DNA ligase 
| T3 DNA ligase 
| 1654 
| 5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.14 / 39350.70 
|}

 

== '''Protein Purification''' ==

1. Step gradient on 10 ml HisTrap column

Buffer A: 10 mM imidazole, 20 mM Tris-HCl pH 8.0 , 500 mM NaCl, 0.05 mM EDTA, 1 mM beta-ME Buffer B: 500 mM imidazole, 20 mM Tris-HCl pH 8, 300 mM NaCl, 0.05 mM EDTA, 1 mM beta-ME method 64 

SDS-PAGE: 2013/4/11

[[Image:1458 1654 1697after first purification.jpg]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| colspan="2" | 1 
| 2 
| colspan="2" | 3 
| colspan="2" | 4 
| 5    
| 6    
|  7   
|  8    
| 10 
| 11 
| 12 
|-
| clone ID
| colspan="5" | 1458 
| colspan="2" | Marker 
| colspan="4" | 1654 
| colspan="4" | 1697 
|-
| name 
| colspan="5" | pT7A3-KOD 
| colspan="2" | 
| colspan="4" | pT7A3-T3 DNA ligase 
| colspan="4" | pCold-GST-(3c)-wt3C 
|-
| host 
| colspan="5" | Nico21DE3plysN 
| colspan="2" | 
| colspan="4" | Nico21DE3plysN 
| colspan="4" | Nico21DE3plysN 
|-
| induction 
| colspan="5" | 0.2 mM IPTG 25oC o/n 
| colspan="2" | 
| colspan="4" |
0.2 mM IPTG 25oC o/n

 

| colspan="4" | 0.2 mM IPTG 25oC o/n 
|-
| purification 
| colspan="5" | 10ml HisTrap HP 
| colspan="2" | 
| colspan="4" | 10ml HisTrap HP 
| colspan="4" | 10ml HisTrap HP 
|-
| MW 
| colspan="5" | 7.53 / 90022.42 
| colspan="2" | 
| colspan="4" | 5.14 / 39350.70 
| colspan="4" | 21KD/8.47 
|}

Combine fraction 31-68, concentrate to 20ml 

2. Gel filtration on TSK 250 gel filtration.

Buffer: 50 mM Na-Phosphate, pH 7.4, 200 mM NaCl 

SDS-PAGE: 2013-4-17

[[Image:1458after2ndNi 1654aftergel filtration 1711 1712small.jpg]] 

{| width="685" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| colspan="2" | 1 
| 2 
| 3 
| 4 
| 5 
| colspan="2" | 6 
|   7   
|    8  
|     9 
|   10  
| colspan="2" | 11 
| 12 
|-
| clone ID
| colspan="6" | 1458 
| colspan="2" | Marker 
| colspan="4" | 1654                                
| colspan="2" |
1711                     

| colspan="2" |
 1712   

|-
| name 
| colspan="6" | pT7A3-KOD 
| colspan="2" | 
| colspan="4" | pT7A3-T3 DNA  ligase 
| colspan="2" | pET28b-hINFA2 h 
| colspan="2" | pLambda-hINFA2 
|-
| host 
| colspan="6" | Nico21DE3plysN 
| colspan="2" | 
| colspan="4" | Nico21DE3plysN 
| colspan="2" | 
| colspan="2" | 
|-
| induction 
| colspan="6" | 0.2 mM IPTG 25oC o/n 
| colspan="2" | 
| colspan="4" |
0.2 mM IPTG 25oC o/n 

| colspan="2" | 
| colspan="2" | 
|-
| purification 
| colspan="6" | 10ml HisTrap HP(2nd time) 
| colspan="2" | 
| colspan="4" | Bio-Sil TSK 250 
| colspan="2" | 
| colspan="2" | 
|-
| MW 
| colspan="6" | 7.53 / 90022.42 
| colspan="2" | 
| colspan="4" | 5.14 / 39350.70 
| colspan="2" | 
| colspan="2" | 
|}

concentrate fraction 28 ~31using spin concentator

3. Dialyze twice overnight. 

Dialysis Buffer:20 mM Tris-HCl pH7.5, 50 mM NaCl, 50% Glycerol , 0.1 mM EDTA 

4.  Store in a buffer (20 mM Tris-HCl pH7.5, 50 mM NaCl, 50% Glycerol, 0.1 mM EDTA, 1mM DTT) at -80oC (T7ligase box). 

Concentration : 2 mg/ml 

[[Category:Cloning]]

1654 pT7A3-T3 DNA ligase - Revision history

Paul.xu at 19:47, 22 April 2013