Excellgen at 20:39, 20 April 2015

2015-04-20T20:39:08Z

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== '''Cloning of 1513-pT7A3-polyAPol''' ==

'''Purpose:''' Cloning of 11513-pT7A3-polyAPol

'''Date: '''2013-2-25---2013-2-

'''Notebook:0'''

'''Page:'''

'''Clone ID:1513'''

'''Experimenters:''' Shanyi

== '''Results''' ==

{| style="width: 281px; height: 66px" border="1" cellpadding="1" cellspacing="1"
|-
| 
| band 1 
| band 3 
|-
| 
| 1279pT7A3 
| 1140 pTXB1-polyAPol 
|-
| enzymes 
| colspan="2" |            Nde1+ Xho1 
|}

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

=== Colony Screening ===

Date: 2013-2-

Make miniprep and digest with Nde1+ Xho1

[[File:1513digest.JPG|RTENOTITLE]]

chose 1513-2 to do protein expression screening ,cannot express.

[[File:1513-1DIGEST.JPG|RTENOTITLE]][[File:1513-1digest.JPG|RTENOTITLE]]

== subcloning ==

subclone polyAPol from 1140 pTXB1-polyAPol to 1279 pT7A3 using Nco1+ Xho1

http://52.1.104.161/eln/images/4/4c/Digest_for_1513.JPG

== Subcloning and Screening ==

Digested by Nde1 and Xho1

[[File:1944 1513 digestion.JPG]]

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

*&lt;br&gt;

== '''Conclusion''' ==

== Protein Expression ==

2013/3/11 

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

{| 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 ==

2013/3/29 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 
|}

 

[[Category:Cloning]]

1513-pT7A3-polyAPol - Revision history

Excellgen at 20:39, 20 April 2015