Editing 1624-pCold-6His-GST-(E)-(3C)

== '''Cloning of 1624-pCold-6His-GST-(E)-(3C)'''  ==

'''Purpose:''' Cloning of 1624-pCold-6His-GST-(E)-(3C) 

'''Date: '''2013-2-5----2013-2-7 

'''Notebook:0''' 

'''Page:''' 

'''Clone ID:1624''' 

'''Experimenters:''' yingying 

== '''Results'''  ==

=== cloning  ===

[[Image:1584+1656+1624 clone 2-5-2013.JPG]]<br> 

Digest 1612 pCold-GST-E with Nde1 + Nco1--&gt; vector<br>ligate to annealed oligos<br>Nde1-3C-Nco1-T+Nde1-3C-Nco1-B 

<br> 

<br> 

=== Colony Screening  ===

Date: 2013-2-7 

Make miniprep and digest with Pst1 + BglII 

[[Image:1584+1624 digest detect 2-7-2013.JPG]] 

<br> 

2013-2-27<br> 

[[Image:1624 1digest detect 2-27-2013.JPG]][[Image:1624 2digest detect 2-27-2013.JPG]] 

2013-3-4 

[[Image:1624 digest detect 3-4-2013.JPG]] 

two Nhe1 sites 

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

== '''Conclusion'''  ==

== '''protein purification'''  ==

[[Image:626+1717+1719+1720 protein screening 5-2-2013.JPG]] 

{| border="1" width="200" cellspacing="1" cellpadding="1"
|-
| lane<br> 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8<br> 
| 9<br> 
| 10<br>
|-
| clone ID 
| <br>626,pT7A3-Taq_d289 
| 
1717,<br> 

pT7A3-6H-(TEV)-MBP-(3C) 

| 
1719, 

pT7A3-MBP-(E3C)-NRF2<br> 

| 1720,pT7A3-MBP-(E3C)-Keap1<br> 
| 1719, 
pT7A3-MBP-(E3C)-NRF2,<br> 

fraction 38<br> 

| <br>1719, 
pT7A3-MBP-(E3C)-NRF2, 

fraction 23<br> 

| 
cre, 

3.9ug/ul<br> 

| cre,4/2/2013<br> 
| 1624,pCold-6His-GST-(E)-(3C)<br> 
| <br>
|-
| host<br> 
| <br>Nico21DE3plysN<br> 
| Nico21DE3plysN<br> 
| BL21DE3pRare2pLysS<br> 
| BL21DE3pRare2pLysS<br> 
| BL21DE3pRare2pLysS<br> 
| <br>BL21DE3pRare2pLysS<br> 
| <br> 
| <br> 
| Nico21DE3plysN<br> 
| <br>
|-
| induction<br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| <br> 
| <br> 
| 0.2 mM IPTG 25 oC o/n<br> 
| <br>
|-
| purification<br> 
| Ni beads<br> 
| Ni beads<br> 
| Ni beads<br> 
| Ni beads<br> 
| 5ml HisTrap HP<br> 
| 5ml HisTrap HP<br> 
| <br> 
| <br> 
| Ni beads<br> 
| <br>
|-
| MW<br> 
| 61KD/5.97<br> 
| 49KD/5.46<br> 
| 112KD/4.82<br> 
| 115KD/5.57<br> 
| 112KD/4.82<br> 
| 112KD/4.82<br> 
| <br> 
| <br> 
| 33KD/6.0<br> 
| <br>
|}

[[Category:Cloning]]