1250 pET28b-KOD-2
Cloning of 1250 pET28b-KOD-2
[edit]Purpose: Cloning of 1250 pET28b-KOD-2
Date: July 6, 2012
Notebook:
Page:
Clone ID: 1250
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]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
[edit]Date: July 10, 2012
Only samples 5 and 6 had positive inserts