Paul.xu at 18:25, 1 April 2013

2013-04-01T18:25:58Z

New page

== '''Cloning of 1097-pET28b-T4-G32''' ==

'''Purpose:''' Cloning of 1097-pET28b-T4-G32

'''Date: '''2012-10-5---2012-10-9

'''Notebook:0'''

'''Page:'''

'''Clone ID:1097'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1097 PCR 10-5-2012.JPG]]

PCR using the following primers and T4 phage DNA,

T4_G32-Nde1-F+T4_G32-Xho1-R 

[[Image:559+567+1097+1411 clone 10-9-2012.JPG]] 

clone into Nde1 + Xho1 digested 1027 pET28b-LambdaBet 

 

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=== Colony Screening ===

Date: 2012-10-9

no clonies in plate

 

 

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

*&amp;lt;br&amp;gt;

== '''Conclusion''' ==

&

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== '''Cloning of 1097-pET28b-T4-G32''' ==

'''Purpose:''' Cloning of 1097-pET28b-T4-G32

'''Date: '''2012-10-12---2012-10-15

'''Notebook:0'''

'''Page:'''

'''Clone ID:1097'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1097 PCR 10-5-2012.JPG]]

PCR using the following primers and T4 phage DNA,

T4_G32-Nde1-F+T4_G32-Xho1-R 

 

[[Image:240+1097+1107+1352+1423 clone 10-12-2012.JPG]] 

{| width="377" cellspacing="1" cellpadding="1" border="1"
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| 
| lane 6 
| lane7 
|-
| 
| PCR product 
| 1026-pET28b-LambdaExo 
|-
| enzymes 
| colspan="2" |    Nde1+Xho1 
|}

 

 

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=== Colony Screening ===

Date: 2012-10-15

mini prep and digest with Nde1+Xho1 

[[Image:240+1097+1107 digest detect 10-15-2012.JPG]] 

clone 1,2 need to sequence,

sequence result shows clone1is not the right clone, 1026 contamination

 

 

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

* 

== '''Conclusion''' ==

== '''Cloning of 1097-pET28b-T4-G32''' ==

'''Purpose:''' Cloning of 1097-pET28b-T4-G32

'''Date: '''2012-12-26

'''Notebook:0'''

'''Page:'''

'''Clone ID:1097'''

'''Experimenters:''' Jennifer Truong 

== '''Results''' ==

=== cloning ===

[[Image:1097 Template.JPG]] 

See lane 1

PCR using the following primers and T4 phage DNA,

clone into Nde1 + Xho1 digested 1027 pET28b-LambdaBet [[Image:1097 Digestion.JPG]]

Cut out lane 3 (insert) and 4 (vector) and ligate together overnight

Date: Jan 3, 2012 

 

[[Image:1097 PCR.JPG]] 

See lane 1 and 4 

USING THE FOLLOWING AMOUNT OF TEMPLATE

Lane 1: 1:10 dilution of straight Phage DNA, 1 uL 

Lane 2: 1:10 dilution|}  of straight Phage DNA, 5 uL 

Lane 3: 1:10 dilution of straight Phage DNA, 10 uL 

Lane 4: 5 uL of purified Phage DNA

Lane 5: 1 uL of unpurifed Phage DNA

Lane 6: 3 uL of unpurified Phage DNA 

PCR using the following primers and T4 phage DNA

T4_G32-Nde1-F acgata CATATG atgtttaaacgtaaatctactgc T4_G32-Xho1-R (gatgaccttttgaatgacctttaaCTCGAG cgagca) tgctcgCTCGAGttaaaggtcattcaaaaggtcatc 

Clone into Nde1 + Xho1 digested 1279 pT7A3 

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=== Colony Screening ===

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

 

== '''Conclusion''' ==

 

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== '''Cloning of 1097 pT7A3-T4-G32''' ==

'''Purpose:''' Cloning of 1097 pT7A3-T4-G322

'''Date: '''2013-1-5--

'''Notebook:0'''

'''Page:'''

'''Clone ID:1097'''

'''Experimenters:''' shanyi

== '''Results''' ==

=== cloning ===

PCR 

[[Image:1045 1049 1097 1538PCR.JPG]] 

digest with Nde1 + Xho1(red is right)

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

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

=== [[Image:1279 v16 1036digest for 1049 1097 1529.JPG]] ===

=== Colony Screening ===

[[Image:1097 1127 1530 1279 1533 1568 1590digest.JPG]] 

 

 
== Protein Expression ==

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]]

1097-pET28b-T4-G32 - Revision history

Paul.xu at 18:25, 1 April 2013