Excellgen at 20:29, 30 December 2014

2014-12-30T20:29:33Z

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== '''Cloning of 1810 pT7A30-M1wt-1-165''' ==

'''Purpose:''' Cloning of1810 pT7A30-M1wt-1-165

'''Date: '''07, 30, 2013 

'''Notebook: '''

'''Page:'''

'''Clone ID: 1810'''

'''Experimenters: '''Shanyi

== '''Results''' ==

=== Cloning ===

subclone from subclone from 1234 pET30b-M1wt-1-165 to 1727pT7A30 using Nde1 + Xho1

[[Image:Digest for1810.JPG]] 

===  Colony screen ===

digest with Nde1 + Xho1

[[Image:1810digest.JPG]] 

 

 

== '''Result intepretation/Summary''' ==

== '''Conclusion''' ==

=== '''Purification ''' ===

2014/12/12-2014/12/19 SHANYI

1234 pET30b-M1wt-1-165 

culrured on 12/13/2014 

Tuner(DE3)plysN ( transformed on Dec.12), culture overnight  0.2mM IPTG RT induce 2hour, cell lysed 

lysis buffer PH is 8.0, containing 10mM Imidazole 

[[Image:2379 1352 1234purification.jpg]] 

lane5-12: 1234 M1wt1-165 eluted from Ni column (elution buffer:25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,) 

combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff 

connect to FPLC, wash column with 20mM Tris-Hcl PH8.0 50mM NaCL, then elute with 50-500ml Nacl PH8.0, finally use 1M Nacl to elute protein

[[Image:2379 1234 1810purification.jpg]]

lane1: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 25 ==>250mM Nacl 

lane2: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 46==>450mM Nacl

lane3: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 49==>1M Nacl 

lane4: 1234 pET30b-M1wt-1-165, culrured on 12/15/2014, lysate supernatant(Tuner(DE3)plysN , 0.4mM IPTG RT induce O/N, 15.5g/4L) 

lane5: 1810 pT7A30-M1wt-1-165, culrured on 12/15/2014, lysate supernatant(Tuner(DE3)plysN , 0.2mM IPTG RT induce O/N, 15.5g/4L) 

lane6: marker 

load lysate to Ni column,

wash with bufferA, then wash with 35mM Imidazole, elute with elution buffer(25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,) 

[[Image:2379 1234 1810purification2.jpg]] 

lane6: Ni column 10mM Imidazole wash flowthrough 

lane7-8:Ni column 35mM Imidazole wash flowthrough 

lane9-12: protein eluted from Ni column 

combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff

connect to FPLC, wash column with 20mM NaPO4 PH7.0, 50mM Nacl, then elute with 50-500ml Nacl PH8.0, finally use 1M Nacl to elute protein 

[[Image:2379 1234 1810purification3.jpg]]

lane1-3: protein eluted from spff( 12/16/2014, f43,f45,f48)

lane4-8: protein eluted from spff(12/18/2014,f9,f11,f29,f31,f44)

dissolve pellet in 4M urea PH8.0 buffer,load to Ni column

lane10: dissolved protein

lane11: Ni flowthrough

 

'''Purification '''

'''12/26/2014----12/29/2014''' 

1810 pT7A30-M1wt-1-165, Tuner(DE3)plysN,  lot number: 80-1810 6mg, 3mg/ml 

culrured on 12/25/2014 in LB containing NaPO4+Glucose, 30oC grow overnight, OD600 very high, 0.5mM IPTG 30oC induce 5hours, 8L, 60g 

add lysis buffer, freeze and thaw once, 25000rpm 25min, wash pellet with lysis buffer twice 

load supernant to Ni column, wash with lysis buffer 

elute with elution buffer(25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,1mM beta-ME) 

dilute with 5%glycerol, protein aggregated, add H3PO4 until it became clear 

load to 25ml spff 

wash with 100ml 25 mM NaPO4 pH 5.0 , 

elute protein with 0-500 mM NaCL in  25 mM NaPO4 pH 5.0 (linear gredient), then elute protein with 1M Nacl in 25 mM NaPO4 pH 5.0 

last, use 0-1M Nacl in PH8.0 buffer to elute protin 

[[Image:1810purification 2014 12 29.jpg]] 

protein eluted from spff 

lane1-9: protein eluted by 1M Nacl in 25 mM NaPO4 pH 5.0 buffer 

lane11,12: protein eluted by 0-1M Nacl in 25 mM Tris-Hcl  pH 8.0  buffer 

[[Category:Cloning]]

1810 pT7A30-M1wt-1-165 - Revision history

Excellgen at 20:29, 30 December 2014