115 pLVX-TagRFP

115: pLVX-TagRFP

subclone TagRFP from 114 pET28b-TagRFP to 90 pLVX-puro-HA-AKR1C4 using BamH1 + Xba1

8/11/2012

1 to 6 digest with Nde1 + Xba1

1*: pLVX-TagGFP #1 digest with Nde1 + Xho1

Nde1--CMV--Xho1--EcoR1--BamH1--TagGFP--Xba1

8/13/2012

digest with Xho1+ Xba1 or Xho1 + BamH1

1	2	3	4	5	6
90		110		115
pLVX-puro-HA-AKR1C4		pLVX-TagGFP		pLVX-TagRFP
Xho1+ Xba1	Xho1 + BamH1	Xho1+ Xba1	Xho1 + BamH1	Xho1+ Xba1	Xho1 + BamH1

90: there are two Xba1 sites, BamH1 is in the middle of Xba1 sites: Xho1----EcoR1--Xba1 -- BamH1 -- Xba1

There is also one EcoR1 site within HA-AKR1C4, that is why there are two insert bands then it is digest with Xba1 + Xho1

and one insert band when digest with Xho1 + BamH1.

No insert for 110 and 115 because the vector was cut with Xba1 + BamH1, and that is why no insert in lane 4 and 6.

Cloning of 115 pLVX-TagRFP

Purpose: Cloning of 115 pLVX-TagRFP

Date: August 15, 2012

Notebook: 

Page:

Clone ID: 115

Experimenters: Jennifer Truong

Results

Cloning

Date: August 15, 2012

digest 114 pET28b-TagRFP with BamH1 + Xba1 -->> TagRFP insert (lane 3)
Digest vector 153 pLVX-Plus with BamH1 + Xba1 -->> vector (lane 4)

Cut out bands, ligate together, transform

Colony Screening

Date: August 17, 2012

Digested MiniPrep with RE BamH1 + Xba1

Samples 1, 4, 5 and 6 may be correct?\

Digested samples 4, 5, 6 in lanes 4, 5, 6 respectively with RE EcoR1 and Xba1

Date: August 24, 2012

Digested MiniPrep with RE EcoR1 and Xba1

Sent sample 1 to sequencing

8/27/2012

digest miniprep with BamH1 + Xba1, clone $4 and #6 are not good