Editing 1097-pET28b-T4-G32

== '''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<br> 

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

clone into Nde1 + Xho1 digested 1027 pET28b-LambdaBet<br><br> 

<br> 

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=== Colony Screening  ===

Date: 2012-10-9 

no clonies in plate 

<br> 

<br> 

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

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

== '''Conclusion'''  ==

&amp; 

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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<br> 

<br> 

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

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

<br><br> 

<br> 

{| cellspacing="1" cellpadding="1" border="1"
|-
| <br>
|}

=== Colony Screening  ===

Date: 2012-10-15 

mini prep and digest with Nde1+Xho1<br> 

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

clone 1,2 need to sequence, 

sequence result shows clone1is not the right clone, 1026 contamination 

<br> 

<br> 

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

*<br>

== '''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<br> 

== '''Results'''  ==

=== cloning  ===

[[Image:1097 Template.JPG]]<br> 

See lane 1 

PCR using the following primers and T4 phage DNA, 

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

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

Date:&nbsp;Jan 3, 2012<br> 

<br> 

[[Image:1097 PCR.JPG]]<br> 

See lane 1 and 4<br> 

USING THE FOLLOWING AMOUNT OF TEMPLATE 

Lane 1:&nbsp;1:10 dilution of straight Phage DNA, 1 uL<br> 

Lane 2:&nbsp;1:10 dilution|}&nbsp; of straight Phage DNA, 5 uL<br> 

Lane 3:&nbsp;1:10 dilution of straight Phage DNA, 10 uL<br> 

Lane 4:&nbsp;5 uL&nbsp;of purified Phage DNA 

Lane 5:&nbsp;1 uL&nbsp;of unpurifed Phage DNA 

Lane 6:&nbsp;3 uL&nbsp;of unpurified Phage DNA&nbsp; 

PCR using the following primers and T4 phage DNA 

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

Clone into Nde1 + Xho1 digested 1279 pT7A3<br> 

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=== Colony Screening<br>  ===

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

<br> 

== '''Conclusion'''  ==

<br> 

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<br> 

== '''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<br> 

[[Image:1045 1049 1097 1538PCR.JPG]]<br> 

digest with Nde1 + Xho1(red is right) 

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

[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]]<br> 

<br> 

<br> 
== Protein Expression  ==

2013/3/29<br> SHANYI 

[[Image:1097 1138 1458 1494 1505 1513 1558 1562 1573 1593 1654NEW.jpg]]<br> 

{| width="832" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br> 
| Name<br> 
| Protein Name<br> 
| Clone ID<br> 
| Host<br> 
| Induction<br> 
| Purification<br> 
| pI/Mw<br>
|-
| M<br> 
| PageRuler Plus Prestained protein ladder<br> 
| &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|-
| 1<br> 
| pT7A3-T4-G32<br> 
| T4-G32<br> 
| 1097<br> 
| 
5 ml,<br> 

Nico21(DE3)plysN 

| 0.2 mM IPTG&nbsp; RT o/n<br> 
| Ni beads<br> 
| 
4.82 / 33506.43 

|-
| 2<br> 
| pT7A3-MBP-(3c)-MutM<br> 
| MBP-(3c)-MutM<br> 
| 1138<br> 
| 
&nbsp;5 ml,<br> Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
8.68 / 30290.07+43.6 <br> 

|-
| 3<br> 
| pT7A3-KOD<br> 
| KOD<br> 
| 1458<br> 
| 
&nbsp; 5 ml,<br> Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads <br> 
| 
7.53 / 90022.42 

|-
| 4<br> 
| pT7A3-UDG<br> 
| UDG<br> 
| 1494<br> 
| 
&nbsp;5 ml,<br> Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
5.45 / 27174.73 

|-
| 5<br> 
| pT7A3-T7-Endo1<br> 
| T7-Endo1<br> 
| 1505<br> 
| 
&nbsp;5 ml,<br> Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
9.54 / 17171.84 &nbsp;<br> 

|-
| 6<br> 
| pT7A3-polyAPol<br> 
| polyAPol<br> 
| 1513<br> 
| &nbsp; 5 ml, 
Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
9.67 / 53870.91<br> 

|-
| 7<br> 
| pT7A3-T7-Exo<br> 
| T7-Exo<br> 
| 1558<br> 
| &nbsp; 5 ml, 
Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
4.93 / 34502.20

|-
| 8<br> 
| pT7A3-MBP-(3C)-Exo-1<br> 
| MBP-(3C)-Exo-1<br> 
| 1562<br> 
| &nbsp; 5 ml, 
Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
8.80 / 18143.72<br> 

|-
| 9<br> 
| pT7A3-MBP-(3C)-RnaseH<br> 
| MBP-(3C)-RnaseH<br> 
| 1573<br> 
| &nbsp; 5 ml, 
Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
8.43 / <br> 

17596.99 +43.6 

|-
| 10<br> 
| pT7A3-MBP-(3c)-E_coli_Endo IV<br> 
| MBP-(3c)-E_coli_Endo IV<br> 
| 1593<br> 
| &nbsp; 5 ml, 
Nico21(DE3)plysN<br> 

| 0.2mM IPTG RT o/n <br> 
| Ni beads<br> 
| 
5.43 / <br> 

31479.53 +43.6 

|-
| 11<br> 
| pT7A3-T3 DNA ligase<br> 
| T3 DNA ligase<br> 
| 1654<br> 
| 5 ml, 
Nico21(DE3)plysN<br> 

| 0.2 mM IPTG RT o/n<br> 
| Ni beads<br> 
| 5.14 / 39350.70 <br>
|}

<br> 

<br>



[[Category:Cloning]]