Paul.xu at 17:25, 21 March 2013

2013-03-21T17:25:37Z

New page

== '''Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2''' ==

'''Purpose:''' Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2 

'''Date: '''Jan 4, 2013 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1123

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: Jan 4, 2013 

PCR using 1uL of 1:10 dilution of T4 genomic DNA and the following primers 

T4RNA-lig-Nde1-F atcgac CATatg tttaaaaagtatagcagtc

T4RNA-lig-Xho1-R (cttggattgagttggtaagttaaCTCGAG atcgaa) ttcgatCTCGAGttaacttaccaactcaatccaag 

See lane 1

[[Image:1123 1045 Amp.JPG]]

Cut out band, and purify

Digest insert (lane ) and vector (1514 pT7A3-MBP-(3C) lane ) with RE Nde1 and Xho1

Cut out bands, ligate together and transform

 

=== Screening ===

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

== '''Conclusion''' ==

 

 

== '''Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2''' ==

'''Purpose:''' Cloning of 1123 pT7A3-MBP-(3C)-T4-RNA-Lig2 

'''Date: '''Jan 4, 2013 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1123

'''Experimenters: '''Shanyi

== '''Results''' ==

=== Cloning ===

PCR

[[Image:1123 1045pcr.JPG]]

 Cut out band, and purify

Digest insert (lane ) and vector (1514 pT7A3-MBP-(3C) lane ) with RE Nde1 and Xho1

[[Image:1049 1097 1123DIGEST-right.JPG]]4.92 / 24309.87

Cut out bands, ligate together and transform

PCR 

[[Image:1045 1049 1122 1123pcr.JPG]] 

[[Image:Digest for 1044 1049 1122 1123 1 16 2013.JPG]] 

 

=== Screening ===

[[Image:1123 1558digest 1 11 2013.JPG]] 

send 1123-2 to sequeng, it is 1026. send 1123-4 to sequence again. 

[[Image:1122 1123DIGEST.JPG]]

== ==

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

== '''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 +43.6      
|-
| 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 +43.6 
|-
| 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+43.6 
|}

 

 

[[Category:Cloning]]

1123 pT7A3-MBP-(3C)-T4-RNA-Lig2 - Revision history

Paul.xu at 17:25, 21 March 2013