Paul.xu at 19:55, 3 August 2012

2012-08-03T19:55:09Z

New page

== '''Cloning of 1023-pET8XHis-AvPAL''' ==

'''Purpose:''' Cloning of 1023-pET8XHis-AvPAL

'''Date: '''2012-3-22--2012-3-28

'''Notebook:0'''

'''Page:'''

'''Clone ID:1023'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-3-22

AvPAL-1-1  to AvPAL-1-16 

AvPAL-2-1  to AvPAL-2-18 

AvPAL-3-1  to AvPAL-3-14 

PCR 3 fragments and then using Gibson mix to join them

 

Digest:

[[Image:1009+1017+1023+1034 clone 3-27-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
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| band 5 
| band 6 
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| 
| PCR product 
| pET8XHis 286 
|-
| enzymes 
| colspan="2" |        Nco1+ Xho1 
|}

 

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=== Colony Screening ===

Date: 2012-3-26

PCR screen  with T7 F+AvPAL-3-14 

no band

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 1023-pET8XHis-AvPAL''' ==

'''Purpose:''' Cloning of 1023-pET8XHis-AvPAL

'''Date: '''2012-3-30--2012-4-2

'''Notebook:'''

'''Page:'''

'''Clone ID:1023'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-3-22

fragment 1:AvPAL-1-1  to AvPAL-1-16 

fragment 2:AvPAL-2-1  to AvPAL-2-18 

fragment 3:AvPAL-3-1  to AvPAL-3-14 

PCR 3 fragments and then fragment 1+fragment 2+fragment 3 as template 

                                        AvPAL-1-1+AvPAL-3-14 as primers 

[[Image:1023+1048 PCR 3-30-2012.JPG]] 

digest : 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
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| band 
| band 
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| enzymes 
| colspan="2" |       
|}

 

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=== Colony Screening ===

Date: 2012- 4-2 

=== [[Image:1023+1034digest detect 4-2-2012.JPG|363x211px|1023+1034digest detect 4-2-2012.JPG]] ===

no insert

 

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

* 

== '''Conclusion''' ==

 

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== '''Cloning of1023-pET8XHis-AvPAL''' ==

'''Purpose:''' Cloning of 1023-pET8XHis-AvPAL

'''Date: '''2012-4-4--2012-4-6

'''Notebook:'''

'''Page:'''

'''Clone ID:1023'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-4-4 

fragment 1:AvPAL-1-1 to AvPAL-1-16 

fragment 2:AvPAL-2-1 to AvPAL-2-18

fragment 3:AvPAL-3-1 to AvPAL-3-14

PCR 3 fragments and then fragment 1+fragment 2+fragment 3 as template

AvPAL-1-1+AvPAL-3-14 as primers

[[Image:1023+1048 PCR 4-4-2012.JPG]] 

 

 

Digest:

[[Image:1023+1036 clone 4-4-2012.JPG]] 

 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
| 
| band 1 
| band 2 
|-
| 
| PCR product 
| pET8XHis 286 
|-
| enzymes 
| colspan="2" |        Nco1+ Xho1 
|}

 

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|}

=== Colony Screening ===

Date: 2012-4-6

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 1023-pET8XHis-AvPAL''' ==

'''Purpose:''' Cloning of 1023-pET8XHis-AvPAL

'''Date: '''2012-4-4--2012-4-25

'''Notebook:'''

'''Page:'''

'''Clone ID:1023'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

Date:2012-4-4

fragment 1:AvPAL-1-1  to AvPAL-1-16 

fragment 2:AvPAL-2-1  to AvPAL-2-18 

fragment 3:AvPAL-3-1  to AvPAL-3-14 

PCR 3 fragments and then fragment 1+fragment 2+fragment 3 as template 

                                        AvPAL-1-1+AvPAL-3-14 as primers 

[[Image:1023+1048 PCR 4-4-2012.JPG]] 

digest : 

[[Image:1022+1023+1048 clone 4-5-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 298px; height: 82px;"
|-
| 
| band 3 
| band 4 
|-
| 
| PCR product 
| pET8XHis-PKAalpha 
|-
| enzymes 
| colspan="2" |       Nco1+ Xho1 
|}

 

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=== Colony Screening ===

Date: 2012- 4-10 

[[Image:1022+1023 digest detect 4-10-2012.JPG]]

clone 3,5,6,7,8 may be right

[[Image:1023 subclone 4-25-2012.JPG]] 

subclone from 1023-3( insert ) to 1023-8 (vector), digest with Nco1+EcoR1 

 

=== subcloning sequencing ===

=== [[Image:1023 digest detect 4-27-2012.JPG]] ===

=== ===

Date:2012/6/13 

[[Image:1023 clone 6-13-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 317px; height: 64px;"
|-
| lane 
| 1 
| 2 
|-
| plasmid 
| 1236 pCDNA3.1-AvPAL-2 
| 1023-1 
|-
| enzymes 
| colspan="2" | BamH1+EcoR1 
|}

 

6/15/2012

digest miniprep with EcoR1 + Bamh1 

[[Image:1235 1238 1933 digestion 6 15 2012.jpg]]

[[Image:1235 1238 1933 digestion2 6 15 2012.jpg]] 

 

[[Image:1235 1238 1023 6 18 2012.jpg]]

 

2012-6-20

digest detect :BamH1+EcoR1

[[Image:1023+1234 digest detect 6-20-2012.JPG]]

 

2012-6-21 

[[Image:1023 clone 6-21-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 286px; height: 80px;"
|-
| lane 
| 1 
| 2 
|-
| 
| 1236pCDNA3.1-AvPAL-2 
| 1023-1 
|-
| enzymes 
| colspan="2" |      BamH1+EcoR1 
|}

 

2012-6-25 

digest detect:BamH1+EcoR1 

[[Image:1023 digest detect 6-25-2012.JPG]]

2012-8-3

digest with:Nco1+Xho1

[[Image:1023_digest_detect_8-3-2012.JPG]]

clone 1.4.6 right

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

* 

== '''Conclusion''' ==

 

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[[Category:Cloning]]

1023-pET8XHis-AvPAL - Revision history

Paul.xu at 19:55, 3 August 2012