Method: Ligations in Low Melting Temperature Agarose

June 7 1990

Srini Ramachandra

Principle:

Ligation of plasmid vector and insert DNAs is carried out directly in melted agarose gel slices. This method is used for subcloning well defined restriction fragments and is probably not suitable for genomic library construction. The method works for both blunt-end and cohesive termini ligations.

Ligation reactions will take about 30 minutes to set up; the reactions are done overnight.

FMC Seaplaque Low Melting Temperature agarose

1. Run the digest on a Low Melting Temperature agarose (FMC Seaplaque) gel of appropriate concentration (usually 1% to 1.2% depending on the size of fragment of interest). Stain the gel and cut out the fragment/s of interest on the UV Prep box (under long wave UV light to minimize DNA degradation) using a clean razor blade. Leave a small amount of each band in the gel to show where the DNA migrated then photograph the gel. Wear protective glasses while working with UV light.

2. Trim the gel piece/s to minimize the agarose and store in eppendorf tubes at 4 degrees C in the dark.

3. For Ligation: Melt the gel pieces on a 70 degrees C heat block for 10 minutes. Measure the volume of the agarose solution in the tube and calculate the volume that would have approximately 300 ng of DNA.

4. To a sterile eppendorf tube add the following:
   • dephosphorylated plasmid DNA: ~ 100 ng
   • 2X Ligation buffer: 10 ul (made fresh from 10X stock and sterile dH2O)
   • T4 DNA Ligase (1U/ul) 0.5 units: 0.5 ul
   • Mix the contents of the tube well.

5. Add approximately 300 ng of the insert DNA (in Low Melt agarose) in a volume of 10 ul or less to the eppendorf tube and mix the contents quickly with a pipet tip before the agarose solidifies. The concentrations of insert and plasmid DNA given above is an example. Usually an insert : plasmid DNA ratio of 3:1 should be used. Ligations work effectively even for bands that are barely visible on a gel.

6. Incubate the ligation mix in a 15 degrees C waterbath for 12-14 hours preferably overnight.

7. Melt the ligation mix on 70 degrees C heatblock for 10 minutes. Make appropriate dilutions with ice-cold TCM buffer and then use for transformation as described under "Transformation of Plasmids/Cosmids into E. coli".

8. Store rest of the ligation mix at 4 degrees C for later use.

Solutions:

1. 10X Ligation buffer:

                                         Final concentration
   1M Tris.HCl, pH7.5     660 ul                 0.66 M
   1M MgCl2                50 ul                 50 mM
   1M DTT                  50 ul                 50 mM
   100 mM ATP             100 ul                 10 mM
   sterile ddH2O          140 ul 
                         1000 ul  --  store in 100ul aliquots at
                                      -20 degrees C.
   

2. 100mM ATP:

      Dissolve 60mg of ATP in 800ul of sterile 
      ddH2O adjust pH to 7.0 with drops of 0.1N 
      NaOH. Test pH using a pH paper. Adjust 
      the volume to 1.0 ml. Filter sterilize and 
      store at  -80 degrees C.
   

3. TCM buffer:

                   
   			           Final concentration
   1M Tris.HCl, pH7.5    100 ul              10 mM
   1M CaCl2              100 ul              10 mM
   1M MgCl2              100 ul              10 mM
   sterile ddH2O         9.7 ml
                         10.0 ml  -- filter sterilize and store in 
                                     1ml aliquots at -20 degrees C.
   

References:

Struhl K. (1985). "A rapid method for creating recombinant DNA molecules". BioTechniques 3: 452-453.