This is for the insertion of dephosphorylated linkers and is not appropriate for use of phosphorylated linkers. Synthetic oligonucleotides provide a simple way to introduce new sequences into a clone. For example, the P element transformation vector Car20ZT.2 has a synthetic linker that provides an initiation codon for translation of ß-galactosidase and a convenient Sal1 site for inserting promoters upstream of the coding sequence. Using dephosphorylated linkers has the advantage that ligation results in the insertion of only one copy of the linker. In contrast, phosphorylated linkers will often attach as polymers and must be designed so that they can be trimmed to a single copy by cutting with an appropriate restriction enzyme.

1. Cut 0.5 to 3 ug of plasmid to completion with the desired restriction enzyme in a volume of 20 ul. You'll use 0.1 ug in your ligation. Check for complete cutting by comparing 50 ng of cut plasmid to 50 ng of uncut plasmid on a minigel. Uncut molecules efficiently transform bacteria so they will produce an undesirable background. The ends left by the restriction enzyme must be compatible with the ends of the synthetic oligonucleotide.
   
   
2. Phenol extract the plasmid after it has been cut completely. Removal of the restriction enzyme is important if a recognition site exists in the plasmid you are trying to construct, since the enzyme may cut the DNA during the ligation. Add 30 ul of 10 mM Tris pH 8, 1 mM EDTA and 100 ul of leder phenol to the digest. Shake vigorously for 1'. Centrifuge at 20^oC for 5'. The upper phase contains the DNA. Carefully remove the lower phase with a pipette and discard it. Extract a second time with 100 ul of leder phenol. Extract in a similar manner with 100 ul of ether. After centrifuging, the ether layer is on top; it should be removed and discarded in a beaker in the hood. Heat the sample, uncapped, at 37^oC for 5' to evaporate residual ether. Add 5 ul of 3M Na Acetate pH 6 and 125 ul of ethanol (precooled to -20^oC). Mix throughly and chill on ice for 15'. Centrifuge at 4^oC for 15'. Discard the supernatant. Add 100 ul of 75% ethanol (precooled to -20^oC), splash it around, spin at 4^oC for 5' and finally discard the supernatant. Try to remove as much of the supernatant as possible. Air dry the sample for 10'. Dissolve the DNA precipitate in 20 ul of 10 mM Tris pH 8, 1 mM EDTA. Check the DNA concentration by using an ethidium bromide drop assay.
   
   
3.  Combine 100 pmoles of each complementary synthetic oligonucleotide in a volume of 10 ul of 10 mM Tris pH 8, 10 mM MgCl₂. Float the tube in a beaker or tub containing several hundred milliliters of water heated to 70^oC. Allow water to slowly cool to room temperature. This allows the complementary oligonucleotides to anneal. 
   
   
4. Set up a 10 ul ligation reaction that contains 100 ng of plasmid, 10 pmoles of annealed oligonucleotide, 1 mM ATP and ligation buffer diluted to 1X (Check the composition of the 10X ligation buffer; it may already contain ATP in which case the additional 1 mM is unnecessary). Mix the components thoroughly and then add 1 unit of T4 DNA ligase. Ligation will occur between the 5' phosphorylated ends of the plasmid and the 3' end of the synthetic oligonucleotides. The 3' ends of the plasmid and the 5' ends of the oligonucleotides will not ligate since they are both dephosphorylated. 
   
   
5. Incubate the sample at 12^oC for several hours. Overnight ligation is OK but probably not necessary because the oligonucleotides are present in high amounts. 
   
   
6. Following the ligation reaction, add 200 ul of 10 mM Tris pH 8, 1 mM EDTA. Heat the sample to 80^oC for 15'. This causes the oligonucleotide that doesn't ligate to the plasmid to dissociate, leaving sticky ends on the plasmid. 
   
   
7. Rapidly chill the sample on ice to prevent reannealing of the oligonucleotides. Add 10 ul of 100 mM spermine HCl (stored frozen at -20^oC), mix and incubate on ice for 15'. The spermine will selectively precipitate the plasmid leaving the smaller oligonucleotides in solution. 
   
   
8. Centrifuge the sample for 15' at 4^oC. Discard the supernatant.
   
   
9. Dissolve the precipitate in 10 ul of 0.6 M Na Acetate pH 6. Allow 15' with intermittent agitation; triterate occasionally towards the end of the incubation. The high salt is required to dissociate the spermine from the DNA. 
   
   
10. Add 10 ul of water and 50 ul of ethanol (precooled to -20^oC) and mix thoroughly. Incubate on ice for 15', and then centrifuge at 4^oC for 20'. Discard the supernatant. Add 100 ul of 75% ethanol (precooled to -20^oC), splash it around, spin at 4^oC for 5' and finally discard the supernatant. Try to remove as much of the supernatant as possible. Air dry the sample for 10'.
    
    
11. Phosphorylate the 5' ends of the plasmid. Dissolve the DNA precipitate in 8 ul of water. Mix in 1 ul of 10X kinase buffer and 0.5 ul of 10 mM ATP. Add 1 unit of polynucleotide kinase and incubate at 37^oC for 1 hour. 
    
    
12. Heat sample to 65^oC for 10' to inactivate the kinase. 
    
    
13. Add 75 ul of water, 10 ul of 10X ligation buffer and 5 ul of 10 mM ATP (again check to see whether the 10X ligation buffer already contains ATP). Add 1unit of ligase and incubate at 12^oC for several hours; overnight is acceptable. This ligation favors intramolecular ligation because the DNA is dilute. 
    
    
14. Transform competent E.coli with 20 ul of the ligation mix. Screen transformants using the mini alkaline plasmid prep.