1368 pLVX-TX 05-HAwt-TagRFP

Cloning of 1368 pLVX-TX 05-HAwt-TagRFP

Purpose: fix pLVX-TX 05-HAwt-TagRFP TagRFP frameshift

Date: 9/7/2012

Notebook: 

Page:

Clone ID: 1368

Experimenters: Yingying

Results

Cloning

Date: 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

Date:

9/11/2012

Clone 1366-1: TagGFP fix plasmid digestion, looks like ligation did not work with T7 ligase + T4 buffer. plasmid can be digested/linearied with EcoR1 and Xho1, but not BamH1