2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H
Cloning of 2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H
[edit]Purpose: Cloning of2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H-==
Date: 2014-12----
Notebook:0
Page:
Clone ID:2379
Experimenters: shanyi
Results
[edit]CLONING
[edit]subclone Flp from 2203 pCold-MBP-Tat-NLS-iFLP-6H using Nde1 + Xho1 TO 2378 pT7A3-Cre
Colony Screening
[edit]Date: 2014_12
Make miniprep, useNde1 + Xho1 to find positive clone
Purification
[edit]02/02/2015-02/04/2015
sample (15ml from 1L culture)
lane1:RT 0.2mM IPTG Induce overnight,,
lane2:30oC 0.4mM IPTG Induce overnight,,
lane3:37oC 0.3mM IPTG Induce overnight,,
lane4:pellte
load lysate to Ni column, elute, protein expression level is very low
dilute(concentration of Nacl is 30mM), load to spFF, before loading A280=0.26 , after loading A280=0.18, 250ml
50-500 mM Nacl elute, no peak, 1M Nacl elute , small peak, run gel, it is not the cre-flp