Paul.xu at 17:30, 21 March 2013

2013-03-21T17:30:53Z

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== '''Cloning of ‎1572 pT7A3-MBP-(3c)-Endonuclease VIII''' ==

'''Purpose:''' Cloning of 1572 pT7A3-MBP-(3c)-Endonuclease VIII

'''Date: '''2013-2-7---

'''Notebook:0'''

'''Page:'''

'''Clone ID:1572'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== PCR ===

PCR from genomic DNA isolated from Escherichia coli str. K-12 substr. MG1655 

[[Image:1572 1588 1593pcr.JPG]] 

Cut out the band, digest PCR products and 1514 pT7A3-MBP-(3C) with Nde1 + Xho1 

[[Image:1572PCRDIGEST.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
|-
| 
| band 2 
| band 3, 
|-
| 
| pcr 
| 1514 
|-
| enzymes 
| colspan="2" |     Nde1 + Xho1 
|}

 

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

=== Colony Screening ===

Date: 2013-

Make miniprep and digest withNde1 + Xho1

[[Image:1572digest.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 
|}

 

 

[[Category:Cloning]]

1572 pT7A3-MBP-(3c)-Endonuclease VIII - Revision history

Paul.xu at 17:30, 21 March 2013