Editing 373-pAd5-CMV-Dest-Flag

'''The primer-Rs of 373 and 373' are different.'''


'''1. Cloning of pAd5-CMV-Dest-Flag (373)''' 

'''Purpose:''' to clone pAd5-CMV-Dest-Flag by PCR 

'''Date:''' 2011-11-28 to 2011-12- 

'''Notebook:''' Xiaojing Wang 

'''Page: '''004127---004131 

'''Clone ID:''' 373 

'''Experimenters:''' Xiaojing Wang 

'''Results''' 

[[Image:327 326 373 PCR 11.29.2011.JPG]]<br> The right bands show PCR product of 373, 



Primer:

ad-GW-F <br>Kpn1 <br>aactc GGTACC ATCAACAAGTTTGTACAAAAAAG



<br>

ad-GW-Flag-R <br> Xho1 <br>(TCTTGTACAAAGTGGTTGATGATTACAAGGATGACGACGATAAGtgaCTCGAGatcga)



(tcttgtacaaagtggttgatgattacaaggatgacgacgataagtgactcgagatcga)<br>( L Y K V V D D Y K D D D D K - )

<br>tcgatCTCGAGtcaCTTATCGTCGTCATCCTTGTAATCATCAACCACTTTGTACAAGA

<br>

<br> 

Digest result: 

[[Image:327 326 373 PCR digest 11.29.2011 S.JPG|153x269px|327 326 373 PCR digest 11.29.2011 S.JPG]][[Image:327 326 373 vector digest 11.29.2011.JPG|356x270px|327 326 373 vector digest 11.29.2011.JPG]] 

<br> 

digest PCR product and vector with Kpn I+Xho I,run gel 

the left picture shows 373 insert, the right picture shows 373 vector (pAd5-CMV-MCS, 280)&nbsp; 

gel purify, ligate, transform ccdb survival,AMP 

<br> 

Colony Screening 

[[Image:326 373 plasmid digest 12.1.2011.JPG]]<br> 

Select 5 clones to mini-prep, digestd by Kpn I and Xho I. 373-1,-2,-3,-5 may be correct. Control is 373-1.<br> 

pDonr221_EGFP(393) + pAd5-CMV-Dest-Flag(373-1) make gateway by LR Clonase II. 

Transform 393+373-1 by STBL3, Amp. 

Select 4 clones to mini-prep. <br> 

[[Image:373+393 gateway 12.8.2011.jpg]]<br> 

<br> 

373+393 1-4 plasmids are digested by BamHI. Control is 373+393-4. <br> 

[[Image:373+393 digest 12.5.2011.JPG|309x361px|373+393 digest 12.5.2011.JPG]]<br> 

373+393 -1 and -4 may be correct.<br> 



'''2. Cloning of pAd5-CMV-Dest-Flag (373')'''<br>Purpose: to clone pAd5-CMV-Dest-Flag by PCR<br>Date: 2011-12-16 to 2012-1-3<br>Notebook: Xiaojing Wang<br>Page: 004127---004131<br>Clone ID: 373'<br>Experimenters: Xiaojing Wang<br>Results<br>

Primer:<br>ad-GW-F <br>Kpn1 <br>aactc GGTACC ATCAACAAGTTTGTACAAAAAAG

ad-GW-Flag-R2 <br> Nhe Xho1 <br>(cttTCTTGTACAAAGTGGTTGATGATTACAAGGATGACGACGATAAGtga gct agc TCGAGatcga) <br>tcgatCTCGAgctagctcaCTTATCGTCGTCATCCTTGTAATCATCAACCACTTTGTACAAGAaag<br>[[Image:327_326'_373'_pcr_12.16.2011_s.jpg]]



Digest result:

[[Image:455_327_326'_373'_252_PCR_digest_12.20.2011_s.JPG]]

[[Image:252_327_326'_373'_455_vector_digest_12.20.2011'_s.JPG]]

Ligation, Transformation<br>Select 5 clones to mini-prep<br>373' plasmids are digested by Kpn1 + Xho1, 373'-1,-2,-3,-5 plasmids may be correct.

[[Image:326'_373'_digest_12.23.2011_s.JPG]]

pDonr221_EGFP(393) + pAd5-CMV-Dest-Flag(373'-1) make gateway by LR Clonase II.<br>Transform 393+373'-1 by STBL3, Amp.<br>Select 4 clones to mini-prep(i.e.373'+393 1-4 plasmids), then transfect 293T cell line.<br>48 hours after transfection, take pictures. 

[[Image:373'+393 1.3.2012.jpg]]

393+373' 1-4 plasmids are digested by BamHI.

[[Image:326'+393_373'+393_digest_bamh1_1.3.2012.JPG]]<br> 373'+393 -1,-2, -3 and -4 may be correct.<br> 

[[Category:Cloning]]