Paul.xu at 18:55, 10 April 2013

2013-04-10T18:55:14Z

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== '''Cloning of 1707-pT7A3-MBP-(E3C)-p53wt''' ==

'''Purpose:''' Cloning of 1707-pT7A3-MBP-(E3C)-p53wt

'''Date: '''2013-3-20---2013-3-22

'''Notebook:0'''

'''Page:'''

'''Clone ID:1707'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1707 clone 3-20-2013.JPG]]

=== Colony Screening ===

Date: 2013-3-22

Make miniprep and digest with Nhe1 + EcoR1 

[[Image:1637+1707+1710 digest detect 3-22-2013.JPG]] 

 

 

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

*&amp;amp;lt;br&amp;amp;gt;

== '''Conclusion''' ==

== '''Protein screening''' ==

2013/3/30

[[Image:1567 1614 1673 1677 1692 1694 1697 1707 1711 protein screen 3-31-2013.JPG]]

{| border="1" width="637" cellspacing="1" cellpadding="1"
|-
| lane 
| name 
| clone ID 
| host 
| induction 
| colspan="2" | purification 
| MW 
|-
| 1 
| pT7A3-T5-Exo 
| 1567 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 21KD/5.95 
|-
| 2 
| pCold-TF-(3c) 
| 1614 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| Ni beads 
| 55.9KD/5.19
|-
| 3 
| pET28b-Nde1 
| 1673 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 44KD/8.41 
|-
| 4 
| pTCold-MBP-(3C) 
| 1677 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 44KD 
|-
| 5 
| pCold-C(3C)-MBP-6H 
| 1692 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 46KD/5.77 
|-
| 6 
| pT7A3-wtSM2 
| 1694 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads, should use CBD beads 
| 33KD/6.30 
|-
| 7 
| pCold-MBP-(3C)-TEV 
| 1696 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 72KD/6.04 
|-
| 8 
| pCold-GST-(3c)-wt3C 
| 1697 
| 4 ml,NiCO21(DE3)pLysN 
| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 21KD/8.47 
|-
| 9 
| pT7A3-MBP-(E3C)-p53wt 
| 1707 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 88KD/5.76 
|-
| 10 
| pET28b-hINFA2 
| 1711 
| 4 ml,
BL21(DE3)Star, pRare2-pLysS 

| 0.2 mM IPTG 25 oC o/n 
| colspan="2" | Ni beads 
| 21KD/5.95 
|}

 

== '''Protein Purification''' ==

1. Step gradient, linear gradient on 5 ml HisTrap column

SDS-PAGE: 2013/4/5

[[Image:1696 1707 1567 1673 1697 protein 4-5-2013.JPG]]

{| border="1" width="200" cellspacing="1" cellpadding="1"
|-
| lane 
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8 
| 9 
| 10 
|-
| clone ID
| colspan="5" | 1696-pCold-MBP-(3C)-TEV
| colspan="2" | 1707-pCold-MBP-(3C)-TEV
| 1567pT7A3-T5-Exo 
| 1673-pET28b-Nde1 
| 1697-pCold-GST-(3c)-wt3C 
|-
| host 
| colspan="5" | Nico21DE3plysN 
| colspan="2" | BL21DE3pRare2pLysS 
| Nico21DE3plysN 
| Nico21DE3plysN 
| Nico21DE3plysN 
|-
| induction 
| colspan="5" | 0.2 mM IPTG 25 oC o/n 
| colspan="2" | 0.2 mM IPTG 25 oC o/n 
| 0.2 mM IPTG 25 oC o/n 
| 0.2 mM IPTG 25 oC o/n 
| 0.2 mM IPTG 25 oC o/n 
|-
| purification 
| colspan="5" | 5ml HisTrap HP 
| colspan="2" | 5ml HisTrap HP 
| Ni beads 
| Ni beads 
| Ni beads 
|-
| MW 
| colspan="5" | 72KD/6.04 
| colspan="2" | 88KD/5.76 
| 33KD/5.22 
| 44KD/8.41 
| 21KD/8.47 
|}

 

2. Gel filtration on TSK 250 gel filtration.

Buffer:

 

[[Image:2013 4 7 pT7A3 MBP E3C p53 gel filtration.jpg]]

SDS-PAGE: 2013/4/9

Peak 1 and 2: p53-MBP fusion and MBP (fraction 17 to 21)

Peak 3: MBP.

[[Image:1696 1707 purification 4-9-2013.JPG]]

concentrate peak 1 (fraction 17 ~21) using spin concentator

Combine MBP fractions stored in cold room.

3. 3C digestion

4. Load on amylose column. Collect flow through. Concentrate protein, store in a buffer (xxx, 10% glycerol) at -80oC. Lot #:

[[Category:Cloning]]

1707-pT7A3-MBP-(E3C)-p53wt - Revision history

Paul.xu at 18:55, 10 April 2013