Editing 115 pLVX-TagRFP

115: pLVX-TagRFP<br> 

<br> 

subclone TagRFP from 114 pET28b-TagRFP to 90 pLVX-puro-HA-AKR1C4 using BamH1 + Xba1<br> 

8/11/2012 <br> 

1 to 6 digest with Nde1 + Xba1<br> 

1*: pLVX-TagGFP #1 digest with Nde1 + Xho1<br> 

[[Image:110 115 8 11 2012.JPG]]<br> 

<br> 

Nde1--CMV--Xho1--EcoR1--BamH1--TagGFP--Xba1 

<br> 

8/13/2012<br> 

digest with Xho1+ Xba1 or Xho1 + BamH1<br> 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 6<br>
|-
| colspan="2" | 90<br> 
| colspan="2" | 110<br> 
| colspan="2" | 115
|-
| colspan="2" | pLVX-puro-HA-AKR1C4<br> 
| colspan="2" | pLVX-TagGFP<br> 
| colspan="2" | pLVX-TagRFP<br>
|-
| Xho1+ Xba1<br> 
| Xho1 + BamH1<br> 
| Xho1+ Xba1<br> 
| Xho1 + BamH1<br> 
| Xho1+ Xba1<br> 
| Xho1 + BamH1<br>
|}

90: there are two Xba1 sites, BamH1 is in the middle of Xba1 sites: '''Xho1----EcoR1--''Xba1'' -- BamH1 -- '''''<b>Xba1</b>''<br> 

There is also one EcoR1 site within HA-AKR1C4, that is why there are two insert bands then it is digest with Xba1 + Xho1<br> 

and one insert band when digest with Xho1 + BamH1. 

<br> 

No insert for 110 and 115 because the vector was cut with Xba1 + BamH1, and that is why no insert in lane 4 and 6.<br> 

[[Image:110 115 8 13 2012.jpg]]<br> 

<br> 

== '''Cloning of 115 pLVX-TagRFP'''  ==

'''Purpose:''' Cloning of 115 pLVX-TagRFP 

'''Date:&nbsp;'''August 15, 2012<br> 

'''Notebook:&nbsp;''' 

'''Page:''' 

'''Clone ID: '''115 

'''Experimenters: '''Jennifer Truong 

== '''Results'''  ==

=== Cloning<br>  ===

Date:&nbsp;August 15, 2012<br> 

digest 114 pET28b-TagRFP with BamH1 + Xba1 --&gt;&gt; TagRFP insert (lane 3)<br>Digest vector 153 pLVX-Plus with BamH1 + Xba1 --&gt;&gt; vector (lane 4)<br> 

[[Image:115 0815 Digestion.JPG]] 

Cut out bands, ligate together, transform 

=== <u></u>Colony Screening<br>  ===

Date:&nbsp;August 17, 2012<br> 

Digested MiniPrep with RE BamH1 + Xba1 <br> 

[[Image:115 0817 Screening.JPG]] 

Samples 1, 4, 5 and 6 may be correct?\ 

Digested samples 4, 5, 6 in lanes 4, 5, 6 respectively with RE EcoR1 and Xba1 

[[Image:0817 Digestion 2.JPG]] 

Date:&nbsp;August 24, 2012<br> 

Digested MiniPrep with RE EcoR1 and Xba1 <br> 

[[Image:115 Screening.JPG]] 

Sent sample 1 to sequencing 



8/27/2012

digest miniprep with BamH1 + Xba1, clone $4 and #6 are not good

[[Image:115_2012_8_27.jpg]]

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

<br> 

== '''Conclusion'''  ==

<br> 

[[Category:Cloning]]