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Created page with "=== Protocol === #Determine the amount of plasmid and insert to be used through the equation X ng plasmid/[(1 M) X bp] = X ng insert/[(X M) X bp]. It is best to have a pasmid..."

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=== Protocol ===

#Determine the amount of plasmid and insert to be used through the equation X ng plasmid/[(1 M) X bp] = X ng insert/[(X M) X bp]. It is best to have a pasmid to insert ratio between 1:4 and 1:10. Note: a useful website for this calculation is http://www.insilico.uni-duesseldorf.de/Lig_Input.html. Also, using 150 ng of vector is a good starting point. 
#Add the following volumes to a round-lidded PCR tube: 

{| cellspacing="1" cellpadding="1" border="1" style="width: 297px; height: 234px;"
|-
! scope="col" | Reagent 
! scope="col" | Volume (μL) 
|-
| DNA 
|
X of insert

X of plasmid 

|-
| 5x T4 DNA ligase buffer 
| 4 
|-
| T4 DNA ligase 
| 1 
|-
| ddH2O 
| up to 20 μL 
|-
| Total 
| 20 
|}

Note: Larger total volumes can be used; however, it is best to keep the total volume as small as possible, without going below 20 μL. 

*Gently mix by tapping the side of the tube.
*Centrifuge the tube for 5 seconds at 4,500 xg
*Leave tube at room temperature overnight.

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Admin: Created page with "=== Protocol === #Determine the amount of plasmid and insert to be used through the equation X ng plasmid/[(1 M) X bp] = X ng insert/[(X M) X bp]. It is best to have a pasmid..."