Editing 1392 pAAV-VP16-EBNA1c (section)

=== Cloning  ===

Date: <br> 

Gene synthesis this VP16 fragment<br> 

[[Image:1298+1348p1+1350 PCR 8-31-2012.JPG]]<br> 

<br> 

PCR out EBNA1C from 371 pCEP4 E02S CK2M EN2L, ~950 bp<br> 

[[Image:1348 PCR2 2012-9-7.JPG]]<br> 

<br> 

9/7/2012: 

PCR 

[[Image:1368 1366 1348 1346 9 7 2012.jpg]]<br> 

<br> <u>'''9/8/2012 Digestion for cloning'''</u><br> 

'''Lane 2, 4, 6:''' TagGFP amplified with TagGFP-EcoR1-BamH1-F &amp; TaqGFP-R3 using 110 pLVX-TagGFP as template as template<br> 

'''Lane #9''': TagRFP amplified with TagRFP-EcoR1-BamH1-F gtattcgaattcGCGGCCGCggatccGTGTCTAAGGGCGAAGAGCTG<br>ATG AGC GGG GGC GAGGAGCTGTTCGCCGGCATCG 

TagRFP-Xba1-R<br>(GCAAACTGGGGCACAAACTTAA TCTAGA CGATCAA)<br>TTG ATC GTC TAG ATT AAG TTT GTG CCC CAG TTT GC<br> 

'''Lane #10''': PCR product VP16 + EBNA1c has this configuration: EcoR1 ---VP16--BamH1--EBNA1c--Xho1<br> 

'''Lane #12''': A/WSN/33 M15/88E amplified <br>FluASeg-7-NdeI-F<br>actagc CAT ATG CAC CAT CAT CAT CAT CAT agtcttctaaccgaggtc 

FluASeg-7-Xho1-R<br>(catcggtctcataggcaaatgCTCGAG atcaga)<br>tctgatCTCGAGcatttgcctatgagaccgatg<br>using 61 pHW2000-A/WSN/33 M15/88E as template<br> 

[[Image:9 8 2012 digestion.jpg]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 598px; height: 107px;"
|-
| <br> 
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 6<br> 
| 7<br> 
| 8<br> 
| 9<br> 
| 10<br> 
| 11<br> 
| 12<br> 
| 13<br>
|-
| Plasmid ID<br> 
| 1333<br> 
| <br> 
| 1334<br> 
| <br> 
| 1329<br> 
| <br> 
| 1330<br> 
| 1329<br> 
| 1368<br> 
| <br> 
| 254<br> 
| <br> 
| 1036<br>
|-
| Plasmid or DNA Name<br> 
| <br> 
| TagGFP<br> 
| <br> 
| TagGFP<br> 
| <br> 
| TagGFP<br> 
| <br> 
| <br> 
| TagRFP<br> 
| VP16 + EBNA1c<br> 
| <br> 
| A/WSN/33 M15/88E<br> 
| <br>
|-
| Digestion<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| EcoR1+ Xba1<br> 
| BamH1 + Xho1<br> 
| BamH1 + Xho1<br> 
| Nde1 + Xho1<br> 
| Nde1 + Xho1<br>
|}

cut out bands as indicated, purify and ligate using T7 ligase + 10X T4 ligase buffer.<br> 

transform DH5a <br>