1250 pET28b-KOD-2

Cloning of 1250 pET28b-KOD-2

Purpose: Cloning of 1250 pET28b-KOD-2

Date: July 6, 2012

Notebook: 

Page:

Clone ID: 1250

Experimenters: Jennifer Truong

Results

Cloning

Date: July 7, 2012

Digest 480 pET28b-KOD with EcoR1 + Not1, see lane 5

Cut out and purify fragment

PCR this ~120bp fragment out from 480 pET28b-KOD using primer
KOD-EcoR1-F TTGAGA GAATTC TGAGAGCCTTC
KOD-Not1-R GGATCCGCGGCCGCGGTACCAGTTCCCTTCGGCTTCAGCCAAG

Expected size ~120 bp, cut out band, elude with 50 uL water

Digest with EcoR1 + Not1
clone back into 480 pET28b-KOD

Digested pET28b-KOD (lane 6) and digested PCRed pET28b-KOD (lane 5), saved vector from lane 6, but need to rePCR the digested pET28b-KOD
Date: June 9, 2012

Expected size ~120 bp (see lane 4), cut out band, purify

Digest with EcoR1 + Not1
clone back into 480 pET28b-KOD

Digest DNA (lane 1) and vector 480 (lane 2) with EcoR1 + Not1
Clone back into 480 pET28b-KOD

Cut out bands in lanes 1 and 2, purify together and ligate

Transform with Top10 and plate on Kan plate, grow overnight

Colony Screening

Date: July 10, 2012

Only samples 5 and 6 had positive inserts