Paul.xu at 18:30, 3 October 2012

2012-10-03T18:30:09Z

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== '''Cloning of 1264-pCMV5-Fc(3c)-NPR1''' ==

'''Purpose:''' Cloning of 1264-pCMV5-Fc(3c)-NPR1

'''Date: '''2012-7-12---2012-7-12

'''Notebook:0'''

'''Page:'''

'''Clone ID:1264'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1264 PCR 7-12-2012.JPG]] 

PCR:

Template: kinase7901 B07

NPR1-Nhe1-F catgac GCTAGC ATGCCGGGGCCCCGGCGCCCCGCTG

NPR1-<strike>Xho1</strike>-R '''wrong primer''' gctgacCTCGAGCTAGCCTCGGGTGCTACTCCCCCTC 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
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| band 2 
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| colspan="2" |           
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=== Colony Screening ===

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

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== '''Cloning of 1264-pCMV5-Fc(3c)-NPR1''' ==

'''Purpose:''' Cloning of 1264-pCMV5-Fc(3c)-NPR1

'''Date: '''2012-7-27---2102-7-30

'''Notebook:0'''

'''Page:'''

'''Clone ID:1264'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1264+1276+1284 PCR 7-27-2012.JPG]] 

PCR

Template:Kinase 7901  B07 primer:NPR1-Nhe1-F+NPR1-Xba1-R 

 [[Image:1263+1264+1269+1276+1284 clone 7-27-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
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| band 3 
| band 7 
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| PCRproduct 
| pCMV5-Fc(3C) 
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| enzymes 
| colspan="2" |       Nhe1+Xba1     
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=== Colony Screening ===

Date: 2012-7-30

Make miniprep and digest with Nhe1+Xba1 

[[Image:1263+1263+1269 digest detect 7-30-2012.JPG]]

no insert

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 1264-pCMV5-Fc(3c)-NPR1''' ==

'''Purpose:''' Cloning of 1264-pCMV5-Fc(3c)-NPR1

'''Date: '''2012-7-31---2012-8-2

'''Notebook:0'''

'''Page:'''

'''Clone ID:1264'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1263+1264+1269+1276 PCR 7-31-2012.JPG]] 

NPR1-Nhe1-F catgac GCTAGC ATGCCGGGGCCCCGGCGCCCCGCTG

NPR1-Xba1-R 

[[Image:1264+1265 clone 7-31-2012.JPG]] 

{| width="345" cellspacing="1" cellpadding="1" border="1"
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| band 1 
| band 2 
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| PCRproduct 
| 1262,pCMV5-Fc(3C) 
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| enzymes 
| colspan="2" |             Nhe1+Xba1 
|}

 

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=== Colony Screening ===

Date: 2012-3-25

Make miniprep and digest with Nco1 + Xba1.

not the right size 

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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== '''Cloning of 1264-pCMV5-Fc(3c)-NPR1''' ==

'''Purpose:''' Cloning of 1264-pCMV5-Fc(3c)-NPR1

'''Date: '''2012-8-3--2012-8-6

'''Notebook:0'''

'''Page:'''

'''Clone ID:1264'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1264+1269+1276+1284 clone 8-3-2012.JPG]][[Image:1263-2 8-3-2012.JPG]] 

{| width="423" cellspacing="1" cellpadding="1" border="1"
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| band 
| band 
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| PCR product 
| 1263-2,pCMV5-Fc(3c)-RIPK3 
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| enzymes 
| colspan="2" |             Nhe+Xba1 
|}

 

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=== Colony Screening ===

Date: 2012-8-6

Make miniprep and digest with Nhe+Xba1

NO insert 

 

c 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 1264-pCMV5-Fc(3c)-NPR1''' ==

'''Purpose:''' Cloning of 1264-pCMV5-Fc(3c)-NPR1

'''Date: '''2012-8-7--2012-8-10

'''Notebook:0'''

'''Page:'''

'''Clone ID:1264'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1264+1276 PCR 8-7-2012.JPG]] 

NPR1-Nhe1-F catgac GCTAGC ATGCCGGGGCCCCGGCGCCCCGCTG

NPR1-Not1-R 

[[Image:1263 clone 8-8-2012.JPG]][[Image:1263 digested 8-8-2012.JPG]] 

 

{| width="423" cellspacing="1" cellpadding="1" border="1"
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| band 
| band 
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| PCR product 
| 1263 pCMV5-Fc(3c)-RIPK3 
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| enzymes 
| colspan="2" |             Nhe1+Not1 
|}

 

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=== Colony Screening ===

Date: 2012-8-10

Make miniprep and digest with Nhe1+Not1

[[Image:1264+1269 digest detect 8-10-2012.JPG]] 

sequencing show mis priming 

  

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 1264-pCMV5-Fc(3c)-NPR1''' ==

'''Purpose:''' Cloning of 1264-pCMV5-Fc(3c)-NPR1

'''Date: '''2012-8-16--2012-8-20

'''Notebook:0'''

'''Page:'''

'''Clone ID:1264'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1264 PCR 8-16-2012.JPG]] 

NPR1-Nhe1-F2 catgac GCTAGC ATGCCGGGGCCCCGGCGCCCCGCTG

NPR1-Not1-R 

[[Image:1224+1264+1276clone 8-17-2012.JPG]] 

 

{| width="423" cellspacing="1" cellpadding="1" border="1"
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| band 3 
| band 4 
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| PCR product 
| 1263 pCMV5-Fc(3c)-RIPK3 
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| enzymes 
| colspan="2" |             Nhe1+Not1 
|}

 

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=== Colony Screening ===

Date: 2012-8-20

Make miniprep and digest with Nhe1+Not1

[[Image:1264+1276 digest detect 8-20-2012.JPG]] 

clone 1,2,3,5,6 may be right

 

 

 

 

  

9/3/2012 

Digest Maxiprep DNA with

Nhe1+ Not1--> GOI

Not1 + Xba1--> to see if SVLT is in the vector

[[Image:9 3 2012.jpg]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
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| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8 
| 9 
| 10 
| 11 
| 12 
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| 1224 
| 1263 
| 1264 
| 1269 
| 1276 
| 1284 
| 1224 
| 1263 
| 1264 
| 1269 
| 1276 
| 1284 
|-
| Nhe1+ Not1 
| Nhe1+ Not1 
| Nhe1+ Not1 
| Nhe1+ Not1 
| Nhe1+ Not1 
| Nhe1+ Not1 
| Not1+ Xba1 
| Not1+ Xba1 
| Not1+ Xba1 
| Not1+ Xba1 
| Not1+ Xba1 
| Not1+ Xba1 
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| lot of RNA
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| lot of RNA
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1224: vector with SVLT, digest with Not1 + Nhe1: why so many bands?? 

1263: why multiple bands with Nhe1 + Not1 digestion? there is an internal Xba1 site, digest with

Not1 + Xba1 --> 500 bp

Transfection: cell aggreggation: 1284 and 1276

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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[[Category:Cloning]]

1264-pCMV5-Fc(3c)-NPR1 - Revision history

Paul.xu at 18:30, 3 October 2012