Paul.xu at 14:18, 6 March 2012

2012-03-06T14:18:33Z

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== pET28b-HTNCre, NiCo21, 37 oC,  4 hours ==

Clone: pET28b-HTNCre, clone # 298 

Cells: NiCo21 transformed with  pET28b-Cre 

Culture conditions: 

Innoculate 1 L LB/K with a single colony of pET28b-HTNCre NiCo21. Grow 8 hours at 37 oC. 

Next day add 100 ml to 1 L LB/K, grow at 37 oC. 

Induction: at OD about 0.8, add 1 ml of 1 M IPTG to 1 L culture, induce at 37 oC for 4 hours 

Cell pellet wet weight:  Some cells appear to be dying after induction,  total 27.4 g 

Lysis: Freeze then thaw one cycle 

Preclear: Homogenize, centrifuge 12000 rpm for 15 min, then 25000 rpm for 20 min. 

Purification Conditions: 10 ml Ni column, step gradient. wash (20 mM imidazole) , elute (300 mM imidazole), collect 3 ml fractions 

Dialysis: 1 X TBS, 50% glycerol, 70 ml, 6 hours, change buffer then dialyse o/n. 

'''Protein''' 

Buffer: 1 X TBS, 50% glycerol 

Lot number on Label: 

Total vol: 

Total amount of protein mg: 

Concentration: 

Size:

Total number of tubes: 

Purity: 

[[Image:Cre recombinase 2 17 2012.jpg]]

 

Samples: 15 ul from  300 mM  imidazole elution fractions (3 ml each) 

MW ladder: 250, 130, 100, 70(red), 55, 35, 25(red), 15, 10(green) 

FERMENTAS PageRulerâ¢ Plus Prestained Protein Ladder 

Gel: Invitrogen NuPAGE Bis-Tris NuPAGE 4-12%, 1.0 mm X 10 wells 

Buffer:  1X Mes-Tris-SDS. (50 mM Mes, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH7.3), diluted from 20 X Mes-SDS-Running buffer from Boston BioProducts 

Stain: Invitrogen Simple Blue 

Destain: water. 

Location: 

== pET28b-HTNCre, NiCo21, 37 oC, 2 hours. ==

Cells: NiCo21 transformed with pET28b-HTNCre. 

Culture conditions:

Innoculate 1 L LB/K/Chl with a single colony of NiCo21 cells from plate. Grow 8 hours.

Next day add 100 ml culture to 1 L LB/K, grow at 37 oC. 

Grow cells in 1 L LB/K/Chl to about OD600 = 0.8 at 37 oC, then cool down to RT. 

Induction: induce at RT with 1 ml of 10% arabinose to 1 L culture and induce overnight 

Pellet: some dead cells (black) Lysis: Freeze and thaw one time

Preclear: Centrifuge 12000 rpm for 15 min, then 25000 rpm for 20 min.

Purification Conditions: 

add 10 ml Ni beads to clarified supernatant, incubate (rotary) for 1 hour in cold room, spin down beads at 1000 rpm for 1 min, remove supernatant, transfer beads to glass column. 

Manual step gradient, wash (20 mM imidazole) , elute (300 mM imidazole), collect 3 ml fractions 

Dialysis: 1 X TBS, 50% glycerol, 70 ml, 6 hours,   change buffer then dialyse o/n. 

'''Protein'''

Buffer: 1 X TBS, 50% glycerol 

Name on Label: 

Lot number on Label:

Storage: 80 oC freezer (storage room) in a freezer box labeled as "HTNCre--Paul, 2/2012" 

Total vol:

Total amount of protein mg:

Concentration:

Size: 

Total number of tubes: 

Purity: 

 

Samples: 15 ul from 300 mM imidazole elution fractions (combined) 

MW ladder: 

ProteinOne

Gel: Invitrogen Bis-Tris NuPAGE 4-12%, 10 wells

Buffer:  

Stain: ProteinOne

Destain: water. 

 

== pET28b-HTNCre, NiCo21, 37 oC,  1.5 hours ==

Clone: pET28b-HTNCre, clone # 298 

Cells:  NiCo21 transformed with  pET28b-Cre 

Culture conditions: 

Innoculate 1 L LB/K/Chl with a single colony of pET28b-HTNCre NiCo21. Grow o/n at 37 oC. 

Next day add 100 ml to 1 L LB/K/Chl, grow at 37 oC. Grow 7 L 

Induction: at OD about 0.8, add 0.5 ml of 1 M IPTG to 1 L culture, induce at 37 oC for 1.5 hours 

Cell pellet wet weight:  cells appear to be healthy,  total 22.4 g (2/14/2012) 

Lysis: Freeze, thaw. 

Preclear: Centrifuge 12000 rpm for 15 min, then 25000 rpm for 20 min. 

Purification Conditions: 

Dialysis: 1 X TBS, 50% glycerol, 70 ml, 6 hours, change buffer then dialyse o/n. 

'''Protein''' 

Buffer: 1 X TBS, 50% glycerol 

Lot number on Label: 

Total vol: 

Total amount of protein mg: 

Concentration: 

Size:

Total number of tubes: 

Purity: Not good, as shown below, probably washing vol is not sufficient. 

 

Samples: 15 ul from  300 mM  imidazole elution fractions (3 ml each) 

MW ladder: 250, 130, 100, 70(red), 55, 35, 25(red), 15, 10(green) 

FERMENTAS PageRulerâ¢ Plus Prestained Protein Ladder 

Gel: Invitrogen NuPAGE Bis-Tris NuPAGE 4-12%, 1.0 mm X 10 wells 

Buffer:  1X Mes-Tris-SDS. (50 mM Mes, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH7.3), diluted from 20 X Mes-SDS-Running buffer from Boston BioProducts 

Stain: Invitrogen Simple Blue 

Destain: water. 

== Buffers ==

Lysis Buffer: 

Wash Buffer: 

Elution Buffer: 

Dialysis Buffer: 

[[Category:Protein_Purification]]

HTNCre Purification Feb 2012 - Revision history

Paul.xu at 14:18, 6 March 2012