Blunt-End Cloning

 

 

1)        Run your vector and insert out on the same agarose gel to estimate relative [DNA] by ethidium bromide staining.  I think this is one of the most critical steps in this procedure---Make sure your starting material is good quality and sufficient quantity. (For cloning, 1 -2 l of your insert or vector should give a clearly visible band on an ethidium bromide-stained gel.)*  I generally like to use 25 - 100 ng of vector and anywhere from 1x to 10x that much insert.  (Most cloning protocols call for a 3:1 molar ratio of insert:vector, but in my experience, the more insert the better!)

 

2)        Prepare ligation reactions in a final volume of 10l.

 

 

#1

#2

#3

#4(optional)

 

(no insert ctrl.)

(low I:V ratio)

(high I:V ratio)

(no ligase ctrl.**)

Insert†

-

2

7

2

Vector†

1

1

1

1

5 mM ATP

1

1

1

1

10x Buffer

1

1

1

1

H2O

6.5

4.5

-

5

Ligase

0.5

0.5

0.5

-

Total volume

10 ml

 

 

 

 

                                                                16 ° C

 


                                                                          4 - 24 hr

 

2)       Transform or store at -20° C.

 

 

 

 

NOTES:

           *If you want to know exact DNA conc., I recommend using a DNA mass ladder (available from BRL), or quantitating by OD260 reading.

 

           ** Ligation #4 is an extra negative control that you should do if think that you might have closed, circular plasmid in either your insert or vector prep. (e.g. If you are using a PCR product and have not gel-purified it from the plasmid template.)

 

           † The actual volumes of insert and vector used will depend on [DNA].