Editing 1682-pCold-6His-GST-(E)-(3C)-NRF2 (section)

== '''Results'''  ==

=== subcloning  ===

1[[Image:1682+1757clone 6-6-2013.JPG]]<br> 

subclone from 1661 pT7A3-MBP-(3c)-NRF2 to 1624 pCold-6His-GST-(E)-(3C) Nde1 + Xho1&lt;--by Yingying<br> 

<br> 

6/7/2013: Digested with Nde1 + Xho1 (done by Yingying)<br> 

[[Image:1682 subcloning.JPG]]<br> 

the following steps were done by Paul 

1: 1661 pT7A3-MBP-(3c)-NRF2<br> 

2: vector 1624 pCold-6His-GST-(E)-(3C)<br> 

gel purification (Qiagen column), elute with 20 ul water. 

ligation, 2.5 ul T4 ligase buffer with 1 ul of T4 and 1 ul T7 DNA ligase, 1 hour, at room temperature<br> 

transform 100 ul NEB Turbo,&nbsp; recovery time 1 hour 

=== Colony Screening  ===

Date: 2013-6-7 

Make miniprep and digest with Nde1 + Xho1&lt;-- By yingying<br> 

<br> 

[[Image:1682 digest 6-7-2013.JPG]] 

Comment by Paul: This gel shows some of the subcloning steps were carelessly done, or were not done properly, most of time this could be caused by rush to do experiment, hoping to "finish" experiment sooner so you can go home erarly or catch bus on time. 

<br> 

The following steps were done by Paul 

6/8/2013<br> 

[[Image:1682 screening.JPG]]<br> 

pick 10 colonies, grow in LB/Kan with 5% glycerol and 2.5% glucose. make minprep, digest with Nde1 + Kpn1<br> 

all clonies are positive, clone #8 vector size is a little bit smaller, do not know why.<br> 

digest colone 1 with three pair of enzymes: <br> 

1: Nde1 + Xho1: good<br> 

2. Nhe1 + Kpn1: good<br> 

3. EcoR1 + Pst1: good<br> 

[[Image:1682 digestion.JPG]]<br> 

Transform BL21Star (DE3)/pRare2pLysS with 0.5 ul of plasmid. Grow o/n and pick a few colonies to grow in 4 L of LB/K/C for protein expression.<br> 

Make glycerol stock of clone #1 in NEB Turbo <br> 

Make glycerol stock of clone # in BL21Star (DE3)/pRare2pLysS<br>