Editing 1063-pLVX-CAG-Dest-IRES-mCherry

Cloning of pLVX-CAG-Dest-IRES-mCherry(1063)<br>Purpose: to clone pLVX-CAG-Dest-IRES-mCherry<br>Date: 2012-4-12 to 2012-4-<br>Notebook: Xiaojing Wang<br>Page: 004224,004215<br> 

Clone ID: 1063<br>Experimenters: Xiaojing Wang<br> 

Results <br> 

PCR gateway cassette using GW-AgeI-F + GW-Flag-R-Xho1 and pVP16,446 as template<br> 

[[Image:PCR gateway cassett 4 17 2012.jpg]]<br> 

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4/17/2012:<br> 

Cut PCR product with Age1 + Xho1<br>Cut vector 1060 pLVX-CAG-MCS-CMV-mCherry with Age1 + Xho1 

[[Image:1063 digestion 4 17 2012.jpg]]<br> 

<br> 

<br>ligate and transform ccdbSurvival<br> 

Select 1 clone to do PCR screening 4/19/2012<br> 

[[Image:1063 205 PCR Screening 4.19.2012.JPG]] 

mini-prep, digest 4/20/2012 

[[Image:1063 205 digest 4.20.2012.JPG]] 

[[Image:1063 digest2 4.20.2012.JPG|83x240px|1063 digest2 4.20.2012.JPG]] 

<br> 

5/1/2012 

Method 2<br>PCR using ccdb-attR1-EcoR1-F &amp; ccdb-attR2-Xho1-R and pVP16,446 as template--reducted ccdb gateway<br> 

[[Image:1062 1063 PCR 5.1.2012.JPG|93x220px|1062 1063 PCR 5.1.2012.JPG]]<br> 

Digest with EcoR1 + Xho1, clone into EcoR1 + Xho1 digested pLVX-CAG-MCS-IRES-mCherry, 1060.<br> 

[[Image:1062 1063 P V digest 5.1.2012.JPG]]<br> 

Ligate and transform ccdb Survival<br> 

Select 4 clones to mini-prep<br>1063 plasmids are digested by EcoR1+Xho1<br> 

[[Image:1062 1063 digest 5.3.2012.JPG]]<br> 

pDonr221_EGFP(393) + 1063-1 make gateway by LR Clonase II. 

Transform 393+1063-1 by STBL3, Amp. 

Select 1 clones to mini-prep. 

1063+393 plasmid is digested by EcoR1+Xho1<br> 

[[Image:1062+393 1063+393 digest 5.8.2012.JPG]]<br> 

<br> 

== '''Cloning of 1063-pLVX-CAG-Dest-IRES-mCherry'''  ==

'''Purpose:''' Cloning of 1063-pLVX-CAG-Dest-IRES-mCherry 

'''Date: '''2012-5-14--- 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:1063''' 

'''Experimenters:''' yingying 

== '''Results'''  ==

=== cloning  ===

[[Image:1063 PCR 5-14-2012.JPG]]<br> 

PCR gateway cassette using GW-AgeI-F + GW-Flag-R-Xho1 and pVP16,446 as template<br> 

[[Image:537+1035+1062+1063clone 5-15-2012.JPG]]<br> 

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| band 5<br> 
| band 6<br>
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| PCR product<br> 
| 1060,pLVX-CAG-MCS-CMV-mCherry<br>
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| enzymes<br> 
| colspan="2" | &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Age1 + Xho1<br>
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=== Colony Screening  ===

Date: 2012-5-17 

Make miniprep and digest with EcoR1+Xho1<br> 

[[Image:1062+1063_digest_detect_5-17-2012.JPG]]

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c <br> 

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

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== '''Conclusion'''  ==

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[[Category:Cloning]]