Editing His-Tat-NLS-Cre-Purification (section)

== Cre activity assay ==

Cre purified with

*1. Ni column only: High activity, high toxicity
*2. Ni column then SP: medium activity, medium toxicity
*3. Ni column then MEP: very low activity, low toxicity.Cre Expression/Induction using Auto-Induce, IPTG and L-arabinose

Clone 1708, and 1010

[[File:Cre expression.jpg|RTENOTITLE]]

Load Cre from 1010 to 20 ml QFF-->50 SP-FF, elute with 300 ml 0f 100 mM-->1M NaCl linear gradient&nbsp;

[[File:Cre 1010 Purification SPFF.jpg|RTENOTITLE]]

Expression screening using 10 ul magnetic beads from expedeon&nbsp;Amintra® Nickel Magnetic Affinity Resin

[[File:Cre375 2603 2642 2643 2644.jpg|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| ID
| Name<br/>
| Host
| MW
| Induction
| Note
| Expression
|-
| 1
| 375
| pET28-Cre<br/>
| Nico21(DE3)pLysN,&nbsp;
| 10.30 / 42.8<br/>
| 1 mM IPTG, 18 oC
| grown on LB/KC, most cells are lysed<br/>
| y
|-
| 2
| 375
| pET28-Cre<br/>
| Rosetta(DE3)
| 10.30 / 42.8<br/>
| 1 mM IPTG<br/>
| looks like contaminated, can not lyse cells
| trace
|-
| 3
| 375
| pET28-Cre
| 
Nico21(DE3)pLysN



| 10.30 / 42.8<br/>
| 1 mM IPTG<br/>
| grown on LB/K<br/>
| y
|-
| 4
| 2603
| pL6H-sso7d-SD-Taq<br/>
| Nico21(DE3)pLysN<br/>
| 6.10 / 104.7<br/>
| 1 mM IPTG<br/>
| <br/>
| y
|-
| 5
| 2642
| pET29b-Flp<br/>
| Nico21(DE3)pLysN<br/>
| 9.76 / 52.5<br/>
| 1 mM IPTG<br/>
| most cells are lysed
| n
|-
| M
| <br/>
| <br/>
| pageruler plus
| <br/>
| <br/>
| <br/>
| <br/>
|-
| 6
| 2643
| pET29b-Int-C3<br/>
| Nico21(DE3)pLysN<br/>
| 9.69 / 41.4<br/>
| 1 mM IPTG<br/>
| <br/>
| maybe
|-
| 7
| 2643<br/>
| pET29b-Int-C3<br/>
| Nico21(DE3)pLysN<br/>
| 9.69 / 41.4<br/>
| 1 mM IPTG<br/>
| <br/>
| maybe
|-
| 8
| 2644
| pET29b-scIHF2<br/>
| Nico21(DE3)pLysN<br/>
| 9.60 / 22.8<br/>
| 1 mM IPTG<br/>
| <br/>
| &nbsp;?
|-
| 9
| 1010
| Cre
| Nico21(DE3)pLysN<br/>
| <br/>
| <br/>
| Lot&nbsp;174-1010
| <br/>
|-
| 10
| 1186
| MBP or MBP-Xis<br/>
| <br/>
| <br/>
| <br/>
| 
| <br/>
|}



[[File:Cre purification magnetic1.jpg|RTENOTITLE]]

Purification of Cre using Ni-NTA mag beads from Genscript and Ni_NTA agarose from GE

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Sample
| <br/>
| <br/>
| <br/>
|-
| 1
| Ni-NTA Mag beads sample 1 frac2
| <br/>
| <br/>
| <br/>
|-
| 2
| Ni-NTA Mag beads&nbsp;sample 1 frac3<br/>
| <br/>
| <br/>
| <br/>
|-
| 3
| Ni-NTA Mag beads sample 1 frac4<br/>
| <br/>
| <br/>
| <br/>
|-
| 4
| Ni-NTA Mag beads sample 2 frac2<br/>
| <br/>
| <br/>
| <br/>
|-
| 5
| Ni-NTA Mag beads&nbsp;sample 2&nbsp;frac3<br/>
| <br/>
| <br/>
| <br/>
|-
| 6
| Ni agarose column&nbsp; 16mm&nbsp; &nbsp; frac 1
| <br/>
| <br/>
| <br/>
|-
| 7
| Ni agarose column&nbsp; 16mm&nbsp; &nbsp; frac 2<br/>
| <br/>
| <br/>
| <br/>
|-
| 8
| Ni agarose column&nbsp; 2.5 cm&nbsp; frac 8<br/>
| <br/>
| <br/>
| <br/>
|-
| 9
| Ni agarose column&nbsp; 2.5 cm&nbsp; frac 11<br/>
| <br/>
| <br/>
| <br/>
|-
| 10
| Ni agarose column&nbsp; 2.5 cm&nbsp; frac 14<br/>
| <br/>
| <br/>
| <br/>
|-
| 11
| Ni agarose column&nbsp; 2.5 cm&nbsp; frac 17<br/>
| <br/>
| <br/>
| <br/>
|-
| M
| pageRuler Plus
| <br/>
| <br/>
| <br/>
|}
[[Category:Protocols]]