Paul.xu at 17:44, 21 March 2013

2013-03-21T17:44:43Z

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== '''Cloning of 1537 pT7A3-Klenow3'-5'exo–''' ==

'''Purpose:''' Cloning of 1537 pT7A3-Klenow3'-5'exo–

'''Date: '''2013-2-4---2013-2-

'''Notebook:0'''

'''Page:'''

'''Clone ID:1537'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== subcloning ===

subclone from 1530pET28b-Klenow(3'->5'_exo–) to 1279 pT7A3 using Nde1+ Sal1

[[Image:DIGEST FOR 1537.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
|-
| 
| band 1 
| band 3 
|-
| 
| 1530 pET28b-Klenow(3'->5'_exo–) 
| 1279pT7A3 
|-
| enzymes 
| colspan="2" |            Nde1+ Sal1 
|}

 

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

=== Colony Screening ===

Date: 2013-2-

Make miniprep and digest with Nde1+ Sal1

[[Image:1537digest 2 19 2013.JPG]][[Image:1537-5-6digest 2 19 2013.JPG]]

[[Image:1537 1558 1570digest 2 20 2013.JPG]]

 

== '''Result intepretation/Summary''' ==

*&lt;br&gt;13-pT7A3-polyAPol

== '''Conclusion''' ==

== Protein Expression ==

2013/3/11 

[[Image:1049 1123 1501 1503 1508 1512 1513 1537 1557 1572.jpg]]13-pT7A3-polyAPol

{| width="832" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Name 
| Protein Name 
| Clone ID 
| Host 
| Induction 
| Purification 
| pI/Mw 
|-
| M 
| Invitrogen SeeBlue plus2 
|                            
| 
| 
| 
| 
| 
|-
| 1 
| pT7A3-T4_RNA_Ligase_1 
| T4_RNA_Ligase_1 
| 1049 
|
5 ml, 

Nico21(DE3)plysN

| 0.2 mM IPTG  RT o/n 
| Ni beads 
| 4.63 / 47485.21 
|-
| 2 
| pT7A3-MBP-(3C)-T4-RNA-Lig2 
| T4-RNA-Lig2 
| 1123 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.11 / 39375.95       
|-
| 3 
| pT7A3-Klenow 
| Klenow 
| 1501 
|
  5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.11 / 71294.91 
|-
| 4 
| pT7A3-T4_RNA_Ligase_ 2_Truncated 
| T4_RNA_Ligase_ 2_Truncated 
| 1503 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.14 / 29824.85 
|-
| 5 
| pT7A3-DNA-Pol-1 
| DNA-Pol-1 
| 1508 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
5.40 / 103118.12 

|-
| 6 
| pT7A3-Phi29DNAPol 
| Phi29DNAPol 
| 1512 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 8.61 / 66714.05 
|-
| 7 
| pT7A3-polyAPol 
| polyAPol 
| 1513 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 9.67 / 53870.91 
|-
| 8 
| pT7A3-Klenow(3'->5'_exo–) 
| Klenow(3'->5'_exo–) 
| 1537 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.77 / 67969.77 
|-
| 9 
| pT7A3-MBP-(3C)-Lambda-Exo 
| Lambda-Exo 
| 1557 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.36 / 25925.62 
|-
| 10 
| pT7A3-MBP-(3c)-Endonuclease VIII 
| Endonuclease VIII 
| 1572 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 7.77 / 29845.13 
|}

 

== Protein Expression 2 ==

2013/3/20 SHANYI

[[Image:1509 1533 1539 1569 1581 1591 1617 1570 1501 1537 1529.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-hTdT 
| hTdT 
| 1509 
|
5 ml, 

Nico21(DE3)plysN

| 0.2 mM IPTG  RT o/n 
| Ni beads 
| 8.66 / 58536.26 
|-
| 2 
| pET28b-T7-DNA-Polymerase 
| T7-DNA-Polymerase 
| 1533 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 6.04 / 79691.65        
|-
| 3 
| pET28b-T7-DNA-Pol-D28 
| T7-DNA-Pol-D28 
| 1539 
|
  5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.90 / 76333.85 
|-
| 4 
| pT7A3-MBP-(3C)-Exo-III 
| MBB-(3C)-Exo-III 
| 1569 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
5.80 / 

30969.17+43.6

|-
| 5 
| pT7A3-T4RNA-Lig2-truncated-KQ 
| T4RNA-Lig2-truncated-KQ 
| 1581 
|
 5 ml, Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
5.14 / 29796.84 

|-
| 6 
| pT7A3-MBP-(3c)-Ecoli-UDG 
| MBP-(3c)-Ecoli-UDG 
| 1591 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
6.67 / 

25693.23 +43.6

|-
| 7 
| pT7A3-MBP-(3c)-newPhi29 
| MBP-(3c)-newPhi29 
| 1617 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
8.61 / 

66714.05 +43.6

|-
| 8 
| pT7A3-MBP-(3C)-Exo-1 
| MBP-(3C)-Exo-1 
| 1570 
|   5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
|
5.51 / 

54484.77 +43.6

|-
| 9 
| pT7A3-Klenow 
| Klenow 
| 1501 
|   5 ml,
Nico21(DE3)plysN 

| 0.1 mM IPTG RT o/n 
| Ni beads 
| 5.08 / 71328.35 
|-
| 10 
| pT7A3-Klenow(3'->5'_exo–) 
| Klenow(3'->5'_exo–) 
| 1537 
|   5 ml,
Nico21(DE3)plysN 

| 0.1 mM IPTG RT o/n 
| Ni beads 
| 5.77 / 67969.77 
|-
| 11 
| pT7A3-Ecoli_RecA 
| Ecoli_RecA 
| 1529 
| 5 ml,
Nico21(DE3)plysN 

| 0.2 mM IPTG RT o/n 
| Ni beads 
| 5.09 / 37973.37 
|}

 

 
 

 

[[Category:Cloning]]

1537 pT7A3-Klenow3'-5'exo– - Revision history

Paul.xu at 17:44, 21 March 2013