Editing 456 pLex-CMV-CuO-Dest

Cloning of pLex-CMV-CuO-Dest (456) 

Purpose: to clone pLex-CMV-CuO-Dest by PCR 

Date: 2011-11-3 to 2011-11- 

Notebook: Xiaojing Wang 

Page: 004113---004114 

Clone ID: 456 

Experimenters: Xiaojing Wang 

Results <br> 

[[Image:456 PCR 11.3.2011.JPG|141x285px|456 PCR 11.3.2011.JPG]]<br> 

The bands show PCR product of 456, 

Primer: 

GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG 

GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG 

Template is 392 pLVB-V5-Dest 

Digest result:&nbsp; <br> 

[[Image:456 PCR digest 11.3.2011.JPG|155x381px|456 PCR digest 11.3.2011.JPG]]<br> 

digest PCR product and vector with Spe1 + Mlu1,run gel 

gel purify, ligate, transform ccdb survival 

<br>Colony Screening <br> 

<br> 

[[Image:456 clone digest 11.11.2011.JPG]] 

Select 5 clones to mini-prep, digestd by Spl I and Mlu I.&nbsp; 456-1,-3.-4 may be correct. <br> 

pDonr221_EGFP(393) +&nbsp;pLex-CMV-CuO-Dest(456-1/456-3/456-4) &nbsp; make gateway by LR Clonase II.<br> 

Transform 393+456-1/456-3/456-4 by STBL3, Amp.<br> 

Select 4 clones each plate to mini-prep. The total number of plasmids is 12 (No.1-12). 

<br> 

[[Image:393+456 gateway by xiaojing 11.18.2011.jpg]] 

No.1-12 plasmids transfect 293T cell line.<br> 

&nbsp;48 hours after transfection, take pictures. 

<br> 

[[Image:393+456 Digest 11.18.2011.JPG]] 

No.1-12 plasmids are digested by BamHI. Bands 13 and 14 are control. 

Band 13 is 456-3 digested by BamHI, and band 14 is 456-3. 

No.1-5,7-9,11,12 may be correct. 

<br> 

Date: 2011-11-7 to 2011-11-10 

Notebook: Yingying Lu<br> 

Page: <br> 

Clone ID: 456 

Experimenters: Yingying Lu<br> 

Results <br> 

[[Image:456 pcr11-7-2011.JPG]]<br> 

GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG 

GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG 

Template is 392 pLVB-V5-Dest <br> 

[[Image:456+493 clone11-8-2011.JPG]]<br> 

digest PCR product and vector448pLex-CMV-CuO with Spe1 + Mlu1,run gel <br> 

band 1 show insert&nbsp; ,band 2 show vector<br> 

[[Image:418+419+456 digest detect11-10-2011.JPG]]<br> 

5 clonies digest detect:Spe1 + Mlu1, 456-3,456-5 may be right<br> 

<br> 

[[Image:393+456 gateway by yingying 11.18.2011.jpg]] 

393EGFP to 456-5&nbsp; 5 clonies&nbsp; transfect into cells <br> 

[[Image:393+456 digest detect11-16-2011.JPG]]<br> 

393-pDonr221_EGFP to 456-5 pLex-CMV-CuO-Dest <br> 

then digest detect with BamH1<br> 

Band 1---5 show 5 clones, band 6 show 456-5 pLex-CMV-CuO-Dest <br> 

<br> 

[[Category:Cloning]]