1392 pAAV-VP16-EBNA1c

Cloning of 1348 pLVX-EF1-VP16-EBNA1c

Purpose: Cloning of 1348 pLVX-EF1-VP16-EBNA1c

Date: 2012-9-7---2012-9-12

Notebook:

Page:

Clone ID: 1348

Experimenters: yingying lu

Results

Cloning

Date:

Gene synthesis this VP16 fragment

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

9/7/2012:

PCR

9/8/2012 Digestion for cloning

Lane 2, 4, 6: TagGFP amplified with TagGFP-EcoR1-BamH1-F & TaqGFP-R3 using 110 pLVX-TagGFP as template as template

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

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

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

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

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

	1	2	3	4	5	6	7	8	9	10	11	12	13
Plasmid ID	1333		1334		1329		1330	1329	1368		254		1036
Plasmid or DNA Name		TagGFP		TagGFP		TagGFP			TagRFP	VP16 + EBNA1c		A/WSN/33 M15/88E
Digestion	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	EcoR1+ Xba1	BamH1 + Xho1	BamH1 + Xho1	Nde1 + Xho1	Nde1 + Xho1

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

transform DH5a

Colony Screening

2012-9-11

BamH1 + Xho1

 no EBNA1c insert

== Cloning of 1399 pAAV--EBNA1c

Purpose: Cloning of 1392 pLVX-EBNA1c

Date: 2012-9-12

Notebook:0

Page:

Clone ID:1392

Experimenters: yingying

Results

cloning

lane 7: EBNA1c PCR product 

lane 8: 254,pAAV.CMV-MCS, pAAV-MCS

BamH1 + Xho1

Colony Screening

Date: 2012-9-14

need to sequence: EBNA1c

pick clone 2  to sequence, right

lane1: VP16 insert

lane2: clone2-1399

EcoR1+BamH1

colony screen

2012-9-19

mini prep and digest detect EcoR1+BamH1 / BamH1 + Xho1

Result intepretation/Summary

• <br>

Conclusion

Note: the cloning vector for assembling VP16-EBNA1c is 254,pAAV.CMV-MCS, pAAV-MCS, not pLVX-EF1. Therefore, need to do subcloning.