Paul.xu at 16:28, 21 February 2013

2013-02-21T16:28:24Z

New page

== '''Cloning of 1613-pET28-cold-GST-3C''' ==

'''Purpose:''' Cloning of 1613-pET28-cold-GST-3C

'''Date: '''2013-1-10---2013-1-14

'''Notebook:0'''

'''Page:'''

'''Clone ID:1613'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1608+1610+1611+1612+1613 pcr 1-10-2013.JPG]] 

[[Image:42+1600+1606+1611 clone 1-11-2013.JPG]] 

 

digest PCR product with Pst1 + Xho1, clone into V20 pET28-cold-GST

{| cellspacing="1" cellpadding="1" border="1"
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| 
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=== Colony Screening ===

Date: 2013-1-14

Make miniprep and digest with Pst1 + Xho1

[[Image:1606+1613 digest detect 1-14-2013.JPG]]

 

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

== '''Conclusion''' ==

== Protein Expression ==

2013/2/12 

Load GST-3C protein (clone: 1613, pET28-cold-GST-3C) purified from Ni column to 1 ml HiTrap GST column, wash with 15 ml of 25 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA. Elute with 10 mM Glutathione in 20 mMTris-HCl, pH7.5, 5 CV, 2 ml per fraction. Use 10 ul for SDS-PAGE 

[[Image:GST-3C purification 2013 2 12.jpg]] 

{| width="381" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Purification 
|-
| M 
| Fermentas 
|-
| 1 
| Eluate from Glutathione beads Fraction 1, 1st column 
|-
| 2 
| Eluate  from Glutathione beads Fraction 2, 1st column 
|-
| 3 
| Eluate from Glutathione beads Fraction 3, 1st column 
|-
| 4 
| Eluate from Glutathione beads Fraction 1, 2nd column 
|-
| 5 
| Eluate  from Glutathione beads  Fraction 2, 2nd column 
|-
| 6 
| Eluate from Glutathione beads Fraction 3, 2nd column 
|-
| 7 
| FT, 1st column 
|-
| 8 
| FT, 2nd column 
|-
| 9 
| wash, 1st column 
|-
| 10 
| load 
|-
| 11 
|
load 

|}

2013/2/13 

Load GST-3C protein (clone: 1613, pET28-cold-GST-3C) purified from Ni column to 1 ml HiTrap GST column, wash with 15 ml of 25 mM Tris-HCl, pH6.9, 600 mM NaCl, 1 mM b-ME, Elute with 10 mM Glutathione in 20 mMTris-HCl, pH7.5, 5 CV, 1 ml per fraction, discard the first fraction (1 ml, no protein). Use 10 ul for SDS-PAGE 

[[Image:GST-3C purification 2013 2 13.jpg]] 

{| width="381" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Purification 
|-
| M 
| Fermentas 
|-
| 1 
| Eluate from Glutathione beads Fraction 1, 1st column 
|-
| 2 
| Eluate  from Glutathione beads Fraction 2, 1st column 
|-
| 3 
| Eluate from Glutathione beads Fraction 3, 1st column 
|-
| 4 
| Eluate from Glutathione beads Fraction 1, 2nd column 
|-
| 5 
| Eluate  from Glutathione beads  Fraction 2, 2nd column 
|-
| 6 
| Eluate from Glutathione beads Fraction 3, 2nd column 
|-
| 7 
| FT, 1st column 
|-
| 8 
| Wash (combined, 30 ul) 
|-
| 9 
| pT7A3-MBP-i3C, 1554, 4 ml ,NiCO21(DE3)pLysN, 0.2 mM IPTG 25 oC o/n, from Ni beads.(only MBP is expressed) 
|-
| 10 
| pT7A3-6His-GST-i3C, 1601, 4 ml, NiCO21(DE3)pLysN, 0.2 mM IPTG 25 oC o/n, from Ni beads.(only GST is expressed)
|}

 

2013/2/21 

[[Image:2_21_2013_protein_purification.jpg]] 

 

{| width="684" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Name 
| Clone ID 
| Host 
| Induction 
| Purification 
|-
| M 
| Invitrogen SeeBlue plus2 
| 
| 
| 
| 
|-
| 1~7 
| pET28-cold-GST-3C 
| 1613 
|
BL21(DE3)Star, pRare2-pLys, 8L 

| 0.2 mM IPTG 25 oC o/n 
| SEC on TSK-Gel 
|-
| 8 
| pT7A3-MBP-(3c)-NRF2 
| 1661 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|-
| 9&11 
| pCold-GST-(3c)-KEAP1 
| 1625 
|
8L

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|}

[[Category:Cloning]]

1613-pET28-cold-GST-3C - Revision history

Paul.xu at 16:28, 21 February 2013