Paul.xu at 18:05, 10 April 2013

2013-04-10T18:05:41Z

New page

== '''Cloning of 1673-pET28b-NdeI''' ==

'''Purpose:''' Cloning of 1673-pET28b-NdeI

'''Date: '''2013-3-4---2013-3-7

'''Notebook:0'''

'''Page:'''

'''Clone ID:1673'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1673pcr1+2 3-4-2013.JPG]] 

 

pcr1+pcr2

[[Image:1673+1677 PCR 3-4-2013.JPG]]

 

[[Image:1542+1662+1673 clone 3-5-2013.JPG]]

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=== Colony Screening ===

Date: 2013-3-7

Make miniprep and digest with Nde1+Xho1 

[[Image:1542+1673+1679 digest 3-7-2013.JPG]]

 

 

 

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

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

== '''Conclusion''' ==

&amp;lt;br&amp;gt;

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== '''Cloning of 1673-pET28b-NdeI''' ==

'''Purpose:''' Cloning of 1673-pET28b-NdeI

'''Date: '''2013-3-11---2013-3-14

'''Notebook:0'''

'''Page:'''

'''Clone ID:1673'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1673pcr1+2+1636pcr.JPG]] 

fix the problem: PCR using Fragment 1 i_NdeI1-1+i_NdeI2-10 

Fragment 2 i_NdeI2-11 + i_NdeI2-14 

Gel purify, then join two fragments using

i_NdeI1-1 +i_NdeI2-14 

 

[[Image:1636+1673+1657 clone 3-12-2013.JPG]] 

 

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=== Colony Screening ===

Date: 2013-3-14

Make miniprep and digest with Nde1+Xho1 

[[Image:1673 digest detect 3-14-2013.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]]

1673-pET28b-NdeI - Revision history

Paul.xu at 18:05, 10 April 2013