Paul.xu at 17:15, 20 July 2012

2012-07-20T17:15:47Z

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== '''Cloning of 537-psPAX2-D64V''' ==

'''Purpose:''' Cloning of 537-psPAX2-D64V by subclone from 514 pLV-Gag-PolD64V to 503 psPAX2 

'''Date: '''2012-1-24--2012-1-26

'''Notebook:'''

'''Page:'''

'''Clone ID:537'''

'''Experimenters:''' yingying

== '''Results''' ==

 

=== Subcloning ===

Date: 2012-1-24

[[Image:537 1st digest 1-24-2012.JPG]] 

 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 426px; height: 80px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 514 pLV-Gag-PolD64V 
| 503 psPAX2 
|-
| digestion enzymes 
| colspan="2" |                Swa1 
|}

[[Image:537 2nd digest 1-25-2012.JPG]] 

 

 

 

=== Colony Screening ===

 

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

* 

== '''Conclusion''' ==

 

=== ===

 

== '''Cloning of 537-psPAX2-D64V''' ==

'''Purpose:''' Cloning of 537-psPAX2-D64V by subclone from 514 pLV-Gag-PolD64V to 503 psPAX2 

'''Date: '''2012-4-5--2012-4-6

'''Notebook:'''

'''Page:'''

'''Clone ID:537'''

'''Experimenters:''' yingying

== '''Results''' ==

 

=== Subcloning ===

Date: 2012-4-4

[[Image:537 clone 4-6-2012.JPG]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 426px; height: 80px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 514 pLV-Gag-PolD64V 
| 503 psPAX2 
|-
| digestion enzymes 
| colspan="2" |                Swa1+AflII 
|}

first digest with  Swa1 for 4 hours at 25c,then digest with AflII for 4 hours at 37c, then run gel ,can not cut out mutated Pol gene fragment from 514 pLV-Gag-PolD64V using Swa1 + AflI 

 

=== Colony Screening ===

 

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

* 

== '''Conclusion''' ==

 

=== ===

 

== '''Cloning of 537-psPAX2-D64V''' ==

'''Purpose:''' Cloning of 537-psPAX2-D64V

'''Date: '''2012-4-16---2012-4-18

'''Notebook:0'''

'''Page:'''

'''Clone ID:537'''

'''Experimenters:''' yingying

== '''Results''' ==

=== subcloning ===

[[Image:537 clone 1st digest 4-13-2012.JPG]] 

first digest with Swa1 for 4 hours at 25c 

[[Image:537+1095 clone 4-16-2012.JPG]] 

2nd digest with AflII for 4 hours at 37c 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 359px; height: 82px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 514 pLV-Gag-PolD64V 
| 503 psPAX2 
|-
| enzymes 
| colspan="2" |             Swa1+AflII. 
|}

 

{| cellspacing="1" cellpadding="1" border="1"
|-
| 
|}

=== Colony Screening ===

Date: 2012-4-18

Make miniprep and sequencing 

no right clone 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 537-psPAX2-D64V''' ==

'''Purpose:''' Cloning of 537-psPAX2-D64V

'''Date: '''2012-5-2---2012-5-4

'''Notebook:0'''

'''Page:'''

'''Clone ID:537'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:537 PCR1+PCR2 5-1-2012.JPG]][[Image:537PCR1 5-2-2012JPG.jpg]][[Image:537 PCR1 5-3-2012.JPG]] 

PCR: PCR1:Int-D64V-F CCAGGAATATGGCAGCTAGTTTG TACACATTTAGAAGG + PgagPol-Seq-R CTTAAAATTAGCAGGAAGATGG

PCR2:pGagPol-Seq-F AGTGATTTTAACCTACCACC +Int-D64V-R CCTTCTAAATGTGTACAAAC TAGCTGCCATATTCCTGG

 PCR1 NO BAND, because of the wrong primer 

 

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=== Colony Screening ===

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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== '''Cloning of 537-psPAX2-D64V''' ==

'''Purpose:''' Cloning of 537-psPAX2-D64V by subclone from 514 pLV-Gag-PolD64V to 503 psPAX2 

'''Date: '''2012-5-9--2012-5-13

'''Notebook:'''

'''Page:'''

'''Clone ID:537'''

'''Experimenters:''' yingying

== '''Results''' ==

 

=== Subcloning ===

Date: 2012-5-9

[[Image:537 PCR1 5-10-2012.JPG]] 

[[Image:537 PCR1+PCR2 5-1-2012.JPG]] 

PCR1:Int-D64V-F CCAGGAATATGGCAGCTAGTTTG TACACATTTAGAAGG +<strike>PgagPol-Seq-R CTTAAAATTAGCAGGAAGATGG </strike>           PgagPol-Seq-R2 ccatcttcctgctaattttaag

 PCR2:pGagPol-Seq-F AGTGATTTTAACCTACCACC +Int-D64V-R CCTTCTAAATGTGTACAAAC TAGCTGCCATATTCCTGG 

[[Image:537 PCR 5-10-2012.JPG]] 

PCR:PCR1+PCR2 as template

     PgagPol-Seq-R2 ccatcttcctgctaattttaag+pGagPol-Seq-F AGTGATTTTAACCTACCACC 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 426px; height: 80px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 
| 
|-
| digestion enzymes 
| colspan="2" |              
|}

 

5/14/2012

514 digestion with different enzymes

[[Image:514 digestion 5 14 2012.jpg]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 515px; height: 82px;"
|-
| Lane
| 1
| 2
| 3
| 4
| 5
| 6
| 7
|-
| Enzyme
| Nde1
| EcoR1
| Nde1+ EcoR1
| Nde1+Sal1
| Nde1+BamH1
| Nde1+ Xho1
| Nde1+AflII
|-
| Fragments
| 2
| 2
| 4
| 2
| 3
| 2
| 2
|}

 

 

=== Colony Screening ===

 

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

* 

== '''Conclusion''' ==

 

=== 2012-5-18----2012-5- ===

[[Image:537 clone 5-18-2012.JPG]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 319px; height: 96px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 514 pLV-Gag-PolD64V 
| 503 psPAX2 
|-
| Enzymes 
| colspan="2" |                            EcoR1 
|}

PCR screen:2012-5-22 

[[Image:537 PCR screen 5-22-2012.JPG]] 

 

2012-5-23

pick 6 clonies for digesttion detect: EcoR1

[[Image:537 digest detect 5-24-2012.JPG]]

no insert

 

 

2012-6-22-----2012-6-28 

[[Image:537 clone 5-18-2012.JPG]] 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| lane 
| 1 
| 2 
|-
| 
| 514 pLV-Gag-PolD64V 
| 503 psPAX2 
|-
| enzymes 
| colspan="2" | EcoR1 
|}

 

2012-6-26

digest detect:

[[Image:537 digest detect 6-26-2012.JPG]]

all have insert

 

2012-6-28

[[Image:537 PCR Screen 6-28-2012.JPG]]

 

== '''Cloning of 537-psPAX2-D64V''' ==

'''Purpose:''' Cloning of 537-psPAX2-D64V

'''Date: '''2012-6-28---2012-7-20

'''Notebook:0'''

'''Page:'''

'''Clone ID:537'''

'''Experimenters:''' yingying

== '''Results''' ==

=== subcloning ===

[[Image:503 514 EcoRV AflII 6 28 2012.jpg]] 

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 281px; height: 66px;"
|-
| 
| band 1 
| band 2 
|-
| 
| 503 psPAX2 
| 514 pLV-Gag-PolD64V 
|-
| enzymes 
| colspan="2" |             EcoRV+AflII 
|}

 

{| cellspacing="1" cellpadding="1" border="1"
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| 
|}

=== Colony Screening ===

Date: 2012-7-5

Make miniprep and digest with EcoR1

[[Image:537 digest detect 7-5-2012.JPG]]

Clone 2,4,5,6 may be right, PCR test

[[Image:537 PCR screen 7-9-2012.JPG]] 

Int-D64V-F CCAGGAATATGGCAGCTAGTTTG TACACATTTAGAAGG + PgagPol-Seq-R2 ccatcttcctgctaattttaag 

clone2,4,5,6 right

2012-7-16-----2012-7-20

pick 8 clones and do miniprep again

digest with EcoR1

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

Clone 2,3,4,5,7,8 may be right

2012-7-20 

PCR screen using pGagPol-Seq-F+pGagPol-Seq-R2 

[[Image:537_PCR_Screen_7-20-2012.JPG]]

choose clone3 PCR product for sequencing

 

 

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

*&lt;br&gt;

== '''Conclusion''' ==

&lt;br&gt;

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&lt;br&gt;

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[[Category:Cloning]]

537-psPAX2-D64V - Revision history

Paul.xu at 17:15, 20 July 2012