Paul.xu at 19:47, 29 May 2013

2013-05-29T19:47:58Z

New page

== '''Cloning of 1625 pT7A3-MBP-(3c)-KEAP1''' ==

'''Purpose:''' Cloning of 1625 pT7A3-MBP-(3c)-KEAP1

'''Date: '''2013-1-24---2013-1- 28

'''Notebook:0'''

'''Page:'''

'''Clone ID:1625'''

'''Experimenters:''' yingying

== '''Results''' ==

=== PCR ===

[[Image:1625 1626 1629 PCR 2013 1 25.JPG]] 

1/27/2013

[[Image:1625 1626 PCR digestion 1 27 2013.jpg]] 

=== Colony Screening ===

Date: 2013-1-

Make miniprep and digest with 

 

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

* 

== '''Conclusion''' ==

 

 

== '''Cloning of 1625 pT7A3-MBP-(3c)-KEAP1''' ==

'''Purpose:''' Cloning of 1625 pT7A3-MBP-(3c)-KEAP1

'''Date: '''2013-1-29---2-13-1-31

'''Notebook:0'''

'''Page:'''

'''Clone ID:1625'''

'''Experimenters:''' yingying

== '''Results''' ==

=== PCR ===

[[Image:1625+1626 pcr 1-29-2013.JPG]] 

[[Image:1625+1626 clone 1-29-2013.JPG]] 

digest pcr product with Nde1 + Xho1, clone into Nde1 + Xho1 digested 1565,pT7A3-MBP-(3C)-sfGFP2 

 

 

=== Colony Screening ===

Date: 2013-1-31

Make miniprep and digest with Nde1 + Xho1 

[[Image:1625+1626 digest detect 1-31-2013.JPG]]

 

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

* 

== '''Conclusion''' ==

 

== Protein Expression ==

2013/2/11 

[[Image:1625 and i3C expression 2013 2 11.jpg]]

 

{| cellspacing="1" cellpadding="1" border="1" width="684"
|-
| Lane 
| Name 
| Clone ID 
| Host 
| Induction 
| Purification 
|-
| M 
| Invitrogen SeeBlue plus2 
| 
| 
| 
| 
|-
| 1 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
|
4 ml, 

BL21(DE3)Star, pRare2-pLysS

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|-
| 2 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|-
| 3 
| pCold-GST-(3c)-KEAP1 
| 1656 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads (wrong, should use Glutathione agarose beads) 
|-
| 4 
| pCold-GST-(3c)-KEAP1 
| 1656 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads (wrong, should use Glutathione agarose beads) 
|-
| 5 
| pET28-cold-NRF2 
| 1626 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|-
| 6 
| pT7A3-MBP-i3C 
| 1554 
| 4 ml,NiCO21(DE3), pRare2-pLysS 
| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|-
| 7 
| pT7A3-6His-GST-i3C 
| 1601 
| 4 ml,NiCO21(DE3), pRare2-pLysS 
| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
|-
| 8 
| 
| 
| 
| 
| 
|}

2/24/2013 

[[Image:2 24 2013 protein purification.jpg]] 

 

{| cellspacing="1" cellpadding="1" border="1" width="684"
|-
| Lane 
| Name 
| Clone ID 
| Host 
| Induction 
| Purification 
|-
| M 
| Invitrogen SeeBlue plus2 
| 
| 
| 
| 
|-
| 1 
| pCold-GST-(3c)-NRF2 
| 1674 
|
4 ml, 

BL21(DE3)Star, pRare2-pLysS

| 0.2 mM IPTG 25 oC o/n 
| Glutathione beads 
|-
| 2 
| pCold-GST-(3c)-NRF2 
| 1674 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Glutathione beads 
|-
| 3 
| pCold-GST-(3c)-NRF2 
| 1674 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Glutathione beads 
|-
| 4 
| pCold-GST-(3c)-NRF2 
| 1674 
|
4 ml,

BL21(DE3)Star, pRare2-pLysS 

| uninduced 
| Glutathione beads 
|-
| 5 
| pET28-cold-GST-3C 
| 1613 
|
8L, BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
|
Ni beads, then gel filtration, Lot # 20130223 

|-
| 6 
| pET28-cold-GST-3C 
| 1613 
| 8L,BL21(DE3)Star, pRare2-pLysS 
| 0.2 mM IPTG 25 oC o/n 
| Ni beads, then gel filtration, Lot # 20130224 
|-
| 7~9 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
| 8L BL21(DE3)Star, pRare2-pLysS 
| 0.2 mM IPTG 25 oC o/n 
| Ni beads, then amylose beads 
|-
| 10~11 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
| 8L BL21(DE3)Star, pRare2-pLysS 
| 0.2 mM IPTG 25 oC o/n 
| Ni beads, then amylose beads 
|}

 2/25/2013 

[[Image:2 25 2013 protein purification.jpg]] 

 

{| cellspacing="1" cellpadding="1" border="1" width="684"
|-
| Lane 
| Name 
| Clone ID 
| Host 
| Induction 
| Purification 
|-
| 1~5 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
|
8L, 

BL21(DE3)Star, pRare2-pLysS

| 0.2 mM IPTG 25 oC o/n 
| Ni beads, amylose beads, gel filtration, fraction # 39~43, this peak has 6His-MBP-KEAP1 fusion. 
|-
| 6 
| Fermentas protein marker 
| 
| 
| 
| 250, 130, 100, 70(red), 55, 35, 25(red), 15, 10 (bottom) 
|-
| 7~12 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
|
8L,

BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads, amylose beads, gel filtration, fraction # 65-70 (peak #2), this is MBP 
|-
| 13 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
|
8L, BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads, amylose beads 
|-
| 14 
| pT7A3-MBP-(3c)-KEAP1 
| 1625 
|
8L, BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| Ni beads, amylose beads, digest with GST-3C protease, Kaep1 without 6His-MBP fusion should be 70KDa (top Red band, fermentas protein marker) 
|-
| 15 
| Invitrogen protein marker 
| 
| 
| 
| 
|}

== Protein expression of KEAP1 clones ==

{| cellspacing="1" cellpadding="1" border="1" width="415" height="227"
|-
| clone ID 
| expression 
| ge image 
| 
|-
| 1625,pT7A3-MBP-(3c)-KEAP1 
| yes 
| [[1625_1626_1656]] 
| 
|-
| 1656,pCold-GST-(3c)-KEAP1 
| need to use GST beads 
| [[1625_1626_1656]] 
| 
|-
| 1676,pCold-MBP-(3C)-KEAP1 
| yes 
| [[1661_1674_1683]] 
| 
|-
| 1701,pMAL-c5E-KEAP1 
| yes 
| 
| 
|-
| 1720,pT7A3-MBP-(E3C)-Keap1 
| no 
| [[626_1717_1719_1720]] 
| 
|-
| 
| 
| 
| 
|}

 

[[Category:Cloning]]

1625 pT7A3-MBP-(3c)-KEAP1 - Revision history

Paul.xu at 19:47, 29 May 2013