Paul.xu at 17:39, 21 March 2013

2013-03-21T17:39:06Z

New page

== '''Cloning of 1539-pET28b-T7-DNA-Pol-D28''' ==

'''Purpose:''' Cloning of 1539-pET28b-T7-DNA-Pol-D28

'''Date: '''2013-2-21----

'''Notebook:0'''

'''Page:'''

'''Clone ID:1539'''

'''Experimenters:''' Shanyi

== '''Results''' ==

=== PCR ===

PCR from 1533

Fragment 1: =>300 bp Fragment 2: =>1700 bp 

[[Image:1535PCR 1539FIRST PCR 2 21 2013.JPG]] 

join 2 fragments by PCR 

[[Image:1539 SENCOND ROUND PCR 2 21 2013.JPG]] 

clone to Nde1 + Xho1  digested pET28b 

[[Image:Idigest for 1535 1539 2 25 2013.JPG]] 

=== Colony Screening ===

Date: 2013-2-

[[Image:1535_1539_1581DIGEST.JPG‎]]

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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== Protein Expression ==

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]]

1539-pET28b-T7-DNA-Pol-D28 - Revision history

Paul.xu at 17:39, 21 March 2013