Paul.xu at 18:03, 10 April 2013

2013-04-10T18:03:24Z

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== '''Cloning of 1614-pCold-TF-(3c)''' ==

'''Purpose:''' Cloning of 1614-pCold-TF-(3c)

'''Date: '''2013-1-28---2013-1-31

'''Notebook:0'''

'''Page:'''

'''Clone ID:1614'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

pcr1 

make promoter by gene synthesis 

pcr2 

PCR TF from Trigger Factor chaperone plasmid 571 pG-Tf2 

8His-TF-F+TF-3C-Nde1-R 

[[Image:1614+1615 pcr1-2 1-28-2013.JPG]]

 

pcr1+pcr2

Join two fragment using Cold-1 + TF-3C-Nde1-R 

[[Image:1611+1614+1615+1629 pcr 1-28-2013.JPG]] 

 

[[Image:1611+1614+1615+1629 clone 1-29-2013.JPG]] 

Digest pcr product with BglII + Nde1.

 Clone into BglII + Nde1 digested v19 pET28-cold 

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=== Colony Screening ===

Date: 2013-1

Make miniprep and digest with  

 

 

 

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

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

== '''Conclusion''' ==

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== '''Cloning of 1614-pCold-TF-(3c)''' ==

'''Purpose:''' Cloning of 1614-pCold-TF-(3c)

'''Date: '''2013-2-1---2013-2-4

'''Notebook:0'''

'''Page:'''

'''Clone ID:1614'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

pcr1 

PCR from v19 pET28-cold using

Cold-BglII-F + 8His-TF-R 

[[Image:1612+1614+1629 pcr1 2-1-2013.JPG]] 

pcr2 

PCR TF from Trigger Factor chaperone plasmid 571 pG-Tf2 

8His-TF-F+TF-3C-Nde1-R 

[[Image:1614+1615 pcr1-2 1-28-2013.JPG]]

 

pcr1+pcr2

Join two fragment using Cold-BglII-F + TF-3C-Nde1-R 

[[Image:1612+1614+1629 pcr 2-1-2013.JPG]] 

 

 

 

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=== Colony Screening ===

Date: 2013-2-4

Make miniprep and digest with  BglII + Nde1 

[[Image:1612+1614+1629 digest detect 2-4-2013.JPG]]

 

 

 

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

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

== '''Conclusion''' ==

&amp;lt;br&amp;gt;

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== '''Cloning of 1614-pCold-TF-(3c)''' ==

'''Purpose:''' Cloning of 1614-pCold-TF-(3c)

'''Date: '''2013-2-14------2013-2-25

'''Notebook:0'''

'''Page:'''

'''Clone ID:1614'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

pcr1 

PCR from v19 pET28-cold using

Cold-BglII-F + 8His-TF-R2 

[[Image:1611+1614 pcr1 2-14-2013.JPG]] 

 

pcr2 

PCR TF from Trigger Factor chaperone plasmid 571 pG-Tf2 

8His-TF-F2+TF-3C-Nde1-R 

[[Image:1611+1614pcr2 2-15-2013.JPG]] 

 

pcr1+pcr2

Join two fragment using Cold-BglII-F + TF-3C-Nde1-R 

[[Image:1614 pcr 2-15-2013.JPG]] 

 

[[Image:336+1614+1676 +1678 clone 2-21-2013.JPG]] 

 

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=== Colony Screening ===

Date: 2013-2-25

Make miniprep and digest with  BglII + Nde1 

[[Image:1614 digest detect 2-25-2013.JPG]] 

 

 

 

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

== '''Conclusion''' ==

== '''protein screening''' ==

[[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 
|}

[[Category:Cloning]]

1614-pCold-TF-(3c) - Revision history

Paul.xu at 18:03, 10 April 2013