375-PET28-Cre
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
band 3,4 show subclone digest:Nco1+Xho1
band 3 show 298 pTriEx-HTNC (Cre) digest result,band 4 show PET28b digest result
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
digest: Nco1+Xho1
mix above three bands insert together,band 2 show vector
5 clonies digest detect:Nco1+Xho1
the later 4 clonies need to sequence for further verification
Expression
[edit]Expression screening using 10 ul magnetic beads from Expedeon
| 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 |
Purification of Cre using Ni-NTA mag beads from Genscript and Ni_NTA agarose from GE
| 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 |
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| 5 | Ni-NTA Mag beads sample 2 frac3 |
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| 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 |
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| 11 | Ni agarose column 2.5 cm frac 17 |
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| M | pageRuler Plus |
| 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
| Lane | Sample | Column/Resin/Beads | |||
| 1 | Cre wash with SP Wash Buffer | SP FF | |||
| 2 | Cre wash with SP Wash Buffer |
SP FF |
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| 3 | Cre wash with SP Wash Buffer |
SP FF |
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| 4 | Cre wash with SP Wash Buffer |
SP FF |
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| 5 | Cre elute with SP FF Elution Buffer | SP FF |
|||
| 6 | Cre elute with SP FF Elution Buffer |
SP FF |
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| 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 |