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Method: Plasmid Subcloning

May 20 1990

Matthew S. Holt


Purpose:

Time required:

Special Reagents:

Preface:

Procedures:

Plasmid Vector Preparation

  1. Digest 10-20 ug of plasmid DNA with the desired restriction enzyme in a total volume of 100 ul. Incubate appropriately for your specific enzyme (usually 37 degrees C) for 1-4 hours. Supercoiled DNA will take longer to cut and it is recommended to use twice as much enzyme.

  2. Dephosphorylate the now linear DNA. Add to the completed digest: 30 ul 10X CIP Buffer 167 ul ddH2O and 3 ul CIP enzyme (1 unit per ul). Incubate at 45 degrees C for 25 minutes.

  3. Prepare an 0.8-1.2% agarose gel with approximately 6 cm wells. This can be achieved by taping several teeth together. Be certain that the taped comb clears the bottom of the gel bed. Also prepare a mini-gel to run a 5 ul aliquot to verifycomplete digestion and determine running time to obtain desired separation.

  4. Add another 3 ul CIP (probably an unnecessary step by CIP specification standards) and incubate at 45 degrees C for another 25 minutes.

  5. Inactivate the CIP enzyme by adding 500 ul of phenol. Vortex then spin in the microfuge at 14000 rpm for 5 minutes. Transfer the aqueous phase to a sterile eppendorf tube.

  6. Add 500 ul of chloroform (to remove phenol) vortex microfuge at 14000 rpm for 2 minutes and transfer the aqueous phase to a sterile tube.

  7. Precipitate the DNA by adding 30 ul 3M NaAcetate pH 5.2 and 800 ul of -20 degrees C EtOH (must be cold!). Microfuge at 4 degrees C immediately for 30 minutes. If you suspect low yields place samples on dry ice for 10 minutes prior to spinning.

  8. Decant the supernatant immediately and vacuum dessicate or lay tube on its side to dry the DNA pellet for 15 minutes. Resuspend in 100 ul 1X glycerol dye and load on gel along with a 1kb ladder (BRL) and several molecular weight standards. Run gel 4-8 hours at 80 volts until the desired separation is obtained.

  9. Stain the gel excise band with a clean razor blade and gel elute the vector (see Gel Elution protocol). Resuspend in 20-50 ul 1X TE and quantitate on a mini-gel. Vector is now ready for ligation.

Insert Preparation

  1. Digest 10-20 ug of insert DNA with the appropriate enzyme in a 100 ul volume. Incubate appropriately (usually 37 degrees C) for a minimum of 4 hours. Remove 5 ul for a test gel.

  2. Prepare an 0.8-1.2% agarose gel with approximately 6 cm wells. This can be achieved by taping several wells together. Be certain that the taped comb clears the bottom of the gel bed. Also prepare a mini-gel to run a 5 ul aliquot to verify complete digestion and running time to obtain desired separation.

  3. After verifying complete digestion add 10 ul of 10X glycerol dye to the digest and load on gel. Run at 80V for 4-8 hours.

  4. Stain gel excise band(s) with a clean razor blade and gel elute the fragments (see Gel Elution protocol). Resuspend in 20-50 ul 1X TE and quantitate in a mini-gel. Inserts are now ready for ligation.

Ligation Reaction

  1. Maniatis has several complicated formulas to determine the amounts of vector and insert to use for the most efficient ligations. To simplify always have a ratio of 3:1 insert to vector. The more DNA used the more efficient the ligation will be. Insert is usually the limiting factor but try to have between 100-300 ng of vector.

  2. Set up the reactions with an appropriate amount of vector and insert 1 ul of 10X Ligation buffer then adjust the volume to 10 ul with ddH2O. Remove 1 ul of the mixture for a negative control then add 1 ul of Ligase (2-4 Weiss units per ul) to the original reaction. Incubate the reaction for a minimum of 4 hours though best results are obtained if allowed to ligate overnight (12 hours). Cohesive-end ligations should be done at 11 degrees C blunt-end ligations at room temperature.

  3. Remove another 1 ul of the ligation mixture to test ligation efficiency: Run the aliquot along with the negative control on a mini-gel. The sample with added ligase will have bands of higher molecular weight if a substantial amount of ligation has occurred.

  4. Use 5 ul of the ligation reaction for transformation (see transformation procedures). The remaining 4 ul can be stored at -20 degrees C and used as a back-up if necessary.
Solutions:

  1. 10X CIP Buffer:
     	10 mM ZnCl2 	10 mM MgCl2 	100 mM Tris pH 8.0  
  2. 10X Ligation Buffer:
      	0.5 M Tris pH 7.4 	0.1 M MgCl2 	0.1 M DTT 	10 mM ATP 	10 mM Spermidine   

References:

Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.53-1.69.