Paul.xu at 18:53, 28 August 2012

2012-08-28T18:53:57Z

New page

== '''Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT''' ==

'''Purpose:''' Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

'''Date: '''2012-7-6---2012-7-16

'''Notebook:0'''

'''Page:'''

'''Clone ID:1224'''

'''Experimenters:''' yingying

== '''Results''' ==

=== subcloning ===

[[Image:1224+1254clone 7-13-2012.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 343px; height: 84px;"
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| band 1 
| band 2 
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| 
| 413 pCDNA3-YFP-IRES-SVLT 
| 1222 pCMV5-Fc(3C)-Dest 
|-
| enzymes 
| colspan="2" |            Not1 + Xba1 
|}

 

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=== Colony Screening ===

Date: 2012-7-16

Make miniprep and digest with Not1 + Xba1 

[[Image:537+1224 digest detect 7-16-2012.JPG]]

clone 3 may be right

clone 3 not the right clone 

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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== '''Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT''' ==

'''Purpose:''' Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

'''Date: '''2012-7-17---2012-7-27

'''Notebook:0'''

'''Page:'''

'''Clone ID:1224'''

'''Experimenters:''' yingying

== '''Results''' ==

=== subcloning ===

[[Image:1224 clone 7-17-2012.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 366px; height: 100px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 413 pCDNA3-YFP-IRES-SVLT 
| 1222-3 pCMV5-Fc(3C)-Dest 
|-
| enzymes 
| colspan="2" |            Not1 + Xba1 
|}

 

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|}

=== Colony Screening ===

Date: 2012-7-19

Make miniprep and digest with Not1 + Xba1 

[[Image:1224 digest detect 7-19-2012.JPG|248x260px|1224 digest detect 7-19-2012.JPG]] 

clone 1,2,3,4,5,6,7,8 may be right

 

digest detect again with :EcoR1+Xba1

[[Image:1224 2digest detect 7-19-2012.JPG|250x266px|1224 2digest detect 7-19-2012.JPG]]

 

2012-7-24 

Gateway 393+1224 digest QC 

[[Image:393+1224 digest 7-24-2012.JPG|181x271px|393+1224 digest 7-24-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 408px; height: 84px;"
|-
| Lane 
| 1 
| 2 
| 3 
| 4 
| 5 
|-
| plasmid 
| colspan="4" | 393 EGFP+1224pCMV5-Fc(3C)-Dest-SVLT 
| 1224pCMV5-Fc(3C)-Dest-SVLT 
|-
| enzymes 
| colspan="5" |                    Nhe+Not
|}

 

plasmid QC: 4 clonies above

[[Image:393+1224-1 plasmid QC 7-24-2012.JPG|182x256px|393+1224-1 plasmid QC 7-24-2012.JPG]]

no lane 

 

2012-7-27

Gateway 393+1224 digest detect :Nhe+Not 

[[Image:393+1224 digest detect 7-27-2012.JPG]] 

clone 3,4,7,8 no insert

 

'''2012-8-6''' 

gateway reaction 393+1224-2 

PCR screen 2012-8-7

Fc-Seq-F+IRES-R-seq 

[[Image:Gateway PCR screen 8-7-2012.JPG]]

lane 1 :positive control 1224-2

 

Gateway reaction

2012-8-9 

[[Image:393+1224+1238 gateway 8-9-2012.JPG]]

1--6 393+1224 6clones ;

7--1224;

8-13 393+1238 6 clones; clone8,10,11,12 transfected into cells,EGFP positive

14--1238; 

[[Image:393+456+1191 8-9-2012.JPG]]

1-6 393+456 6clones; clone1,2,3 transfected into cells,EGFP positive

7--456;

8--13 393+1191 6clones; clone8,9,10,11,12 transfected into cells,EGFP positive

14--1191

== = ==

8/9/2012: 1224 + 393: Digest with Nhe1 + Not1

[[Image:1024 gateway rxt Nhe1 Not1 8 9 2012.jpg]]

2012-8-10 

393+1224 pcr sreenning of purified plasmid DNA: Fc-Seq-F+IRES-R-seq using Phusion (homemade) 

[[Image:393+1224 pcr screen 8-10-2012.JPG]]

lane 1-6: six clones;   lane7: 1224plasmid

8/10/2012 PCR screening of bacterial colonies using Fc-Seq-F+IRES-R-seq and Taq polymerase 

[[Image:393 1224 pcr sreenning 8 11 2012.jpg]] 

c:393+1238

 

1224 digest detect:2012-8-14

[[Image:1224 digest detect 8-14-2012.JPG]]

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

*&lt;br&gt;

== '''Conclusion''' ==

 

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== '''Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT''' ==

'''Purpose:''' Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

'''Date: '''2012-8-22---2012-8-23

'''Notebook:0'''

'''Page:'''

'''Clone ID:1224'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

pick 6 clonies(2012.7.17) for growing 

 

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=== Colony Screening ===

Date: 2012-8-23

Make miniprep and digest with 

[[Image:1224 digest detect 8-23-2012.JPG]] 

clone 6 may be right 

8/24/2012, test plasmid stability

Restransform ccdbsurvial with clone #6 plasmid DNA , pick 8 clones. digest miniprep

with Not1 + Xba1.

[[Image:1224 2012 8 27.jpg]]

2012-8-28

[[Image:1224_digest_detect_8-28-2012.JPG]]

1:Not1+Xba1

2-8:EcoR1+Xba1

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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[[Category:Cloning]]

1224-pCMV5-Fc(3C)-Dest-SVLT - Revision history

Paul.xu at 18:53, 28 August 2012