Paul.xu at 18:32, 20 December 2019

2019-12-20T18:32:07Z

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Cloning of 375-PET28b-Cre

Purpose: to subclone from 298 pTriEx-HTNC (Cre) to PET28b

Date: 2011-10-25----

Notebook:Lu yingying

Page:

Clone ID:375

Experimenters: lu yingying Results

[[File:414+375 subclone digest10-25-2011.JPG|RTENOTITLE]]

band 3,4 show subclone digest:Nco1+Xho1

band 3 show 298 pTriEx-HTNC (Cre) digest result,band 4 show PET28b digest result

[[File:414+470+375+359 digest detect10-27-2011.JPG|RTENOTITLE]]

375 band 1-3 show digest detect result:Nco1+Xho1

Cloning of 375-PET28b-Cre

Purpose: to subclone from 298 pTriEx-HTNC (Cre) to PET28b

Date: 2011-11-4------2011-11-7

Notebook:Lu yingying

Page:003967------3969

Clone ID:375

Experimenters: lu yingying Results

[[File:375 clone -298 insert digest2-11-3-2011.JPG|RTENOTITLE]]

[[File:375+395 clone 11-4-2011.JPG|RTENOTITLE]]

digest: Nco1+Xho1

mix above three bands insert together,band 2 show vector

[[File:359+375 digest detect11-7-2011.JPG|RTENOTITLE]]

5 clonies digest detect:Nco1+Xho1

the later 4 clonies need to sequence for further verification

== Expression ==

Expression screening using 10 ul magnetic beads from Expedeon

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

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

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

[[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
| 
| 
| 
|-
| 1
| Ni-NTA Mag beads sample 1 frac2
| 
| 
| 
|-
| 2
| Ni-NTA Mag beads sample 1 frac3 
| 
| 
| 
|-
| 3
| Ni-NTA Mag beads sample 1 frac4 
| 
| 
| 
|-
| 4
| Ni-NTA Mag beads sample 2 frac2 
| 
| 
| 
|-
| 5
| Ni-NTA Mag beads sample 2 frac3 
| 
| 
| 
|-
| 6
| Ni agarose column  16mm    frac 1
| 
| 
| 
|-
| 7
| Ni agarose column  16mm    frac 2 
| 
| 
| 
|-
| 8
| Ni agarose column  2.5 cm  frac 8 
| 
| 
| 
|-
| 9
| Ni agarose column  2.5 cm  frac 11 
| 
| 
| 
|-
| 10
| Ni agarose column  2.5 cm  frac 14 
| 
| 
| 
|-
| 11
| Ni agarose column  2.5 cm  frac 17 
| 
| 
| 
|-
| M
| pageRuler Plus
| 
| 
| 
|}

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| Ni-NTA 
|}

12/18/19

Purification of Cre using Ni-NTA agarose beads vs Fastflow Ni-NTA

competent cells: Nico21(DE3)

LB with additional 10 g  yeast per L

10 ml 5025

induction: 0.6 mM IPTG 5 hours at 37 oC

Lysis Buffer, 25 mM KPO4, pH 7.4, 500 mM NaCl, 10 mM imidazole, 0.2% NP40

Ni Wash Buffer: the same as Lysis Buffer

Low salt Ni Elution Buffer, 300 mM Imidazole, 25 mM KPO4, pH 7.4, 50 mM NaCl

SP Wash Buffer: 500 mM NaCl, 25 mM KPO4, pH 7.4, 15% Trhalose,  100 mM Arginine, 1 mM beta-ME

SP FF Elution Buffer: use 1.5 M NaCl,  25 mM KPO4, pH 7.4, 15% Trhalose, 100 mM Arginine, 1 mM beta-ME

[[Category:Cloning]][[File:Ni-NTA beads Cre Purification.jpg]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| Sample
| Column/Resin/Beads
| 
| 
| 
|-
| 1
| Cre wash with SP Wash Buffer
| SP FF
| 
| 
| 
|-
| 2
| Cre wash with SP Wash Buffer 
| SP FF 
| 
| 
| 
|-
| 3
| Cre wash with SP Wash Buffer 
| SP FF 
| 
| 
| 
|-
| 4
| Cre wash with SP Wash Buffer 
| SP FF 
| 
| 
| 
|-
| 5
| Cre elute with SP FF Elution Buffer
| SP FF 
| 
| 
| 
|-
| 6
| Cre elute with SP FF Elution Buffer 
| SP FF 
| 
| 
| 
|-
| 7
| Cre Elute with Ni Elution Buffer
| Cube Magnetic NTA-agarose beads
| 
| 
| 
|-
| 8
| Cre Elute with Ni Elution Buffer 
| Cube Magnetic NTA-agarose beads 
| 
| 
| 
|-
| 9
| Cre Elute with Ni Elution Buffer 
| Genscript Magnetic NTA-agarose beads
| 
| 
| 
|-
| 10
| 2648 ADTZ, secreted 
| NTA-agarose beads 
| 
| 
| 
|-
| 11
| 2648 ADTZ, pellet 
| NTA-agarose beads 
| 
| 
| 
|-
| M
| pageruler Plus
| 
| 
| 
| 
|}

375-PET28-Cre - Revision history

Paul.xu at 18:32, 20 December 2019