Editing Gateway-Cloning (section)

== LR Reaction to Transfer Genes to Expression Vector-- for 1 reaction<br>  ==

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

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2. Calculate the volume in ul needed of ENTR plasmid (25 ng)<br>ul needed = 25 ng needed / (concentration in ng/ul)<br> 

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3. Calculate the volume in ul needed of DEST plasmid (25 ng)<br>ul needed = 25 ng needed / (concentration in ng/ul)<br> 

<font color="red">Very Important: never use more than 25 ng Dest vector</font><br> 

4. Prepare in an autoclaved tube<br> 

*ENTR-vector (25 ng)&nbsp; 0.5 ul<br> 
*DEST-vector (<font color="red">25 ng</font>)&nbsp; 0.5 ul

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5. Remove the 5 x LR-Clonase II Enzyme Mix from -20°C or &nbsp;-80°C<br>This is expensive stuff don't leave it to rot in the ice-bucket! 

<br>6. Add '''<font color="red">0.25 ul</font>''' of the 5 x LR-Clonase Enzyme II Mix to the Gateway reaction and mix well 

<br>7. Store the enzyme mix and buffer '''immediately''' at -80°C&nbsp;!!!<br> 

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8. Incubate at '''room temperature''' for<span style="font-weight: bold;"> </span>'''1 to&nbsp; 5 hours or O/N'''.<br> 

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

<br>10. Transform bacteria with entire reaction: 

Mammalian expression vectors such as Adenovirus, Lentivirus and AAV vectors: STBL 3 

Bacterial Expression Vectors: STBL3, Top10 or DH5alpha 

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11. Plate bacteria with proper antibiotic (Amp) selection.<br> <br> 12. Digestion, PCR or sequencing to verify clones 

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