Editing Gateway-Cloning (section)

== LR Reaction to Transfer Genes to Expression Vector-- for 8 reactions<br>  ==

1. Measure the DNA concentration of both constructs (Donor and Destination)<br> 

2.&nbsp; Take out&nbsp; 5 x LR-Clonase II Enzyme Mix from&nbsp; -80°C,&nbsp; put it in dry ice. <br> 

Prepare in an autoclaved tube<br> 

*DEST-vector (200 ng):&nbsp; 4 ul&nbsp; 
*5 x LR-Clonase II Enzyme Mix: 2 ul

mix well by pipetting up and down for a few times 

Note: LR Clonase II is very expensive stuff,&nbsp; don't leave it to rot in the ice-bucket! Put it back to -80 °C immediately.<br> 

3. Aliquot 0.7 ul to 8 tubes.<br> 

4. Add 0.5 ul DONOR or ENTR vector with gene of interest. 

<br> 

5. Incubate at '''room temperature''' for '''5 hours or O/N'''. 

6. Add 0.25 ul of Proteinase K solution and incubate for 10 min at 37°C 

7. Transform bacteria with entire reaction: 

Adenovirus, Lentivirus and AAV vectors: STBL 3 

Bacterial Expression Vectors: Top10 or DH5alpha<br> 

8. Plate bacteria with proper antibiotic (Amp) selection.<br> 9. Digestion or sequencing to verify<br> 

Do not use the following protocol so far.