DNA Ligation Protocol

Material Provided

• 0.5 U/µl T4 DNA igase

• 10x T4 DNA ligase buffer

• restriction digested vector DNA

• restriction digested PCR/insert DNA

Step 1

• thaw T4 DNA ligase buffer at room temp

• assure that all precipitates are re-dissolved prior to use

Step 2

• calculate DNA concentrations and molar ratios for optimal reaction

• we pre reactions of 1:0, 1:1 and 1:3 ratios of plasmid:insert

Step 3

• at room temp, assemble 20µl ligation reactions by adding reagents in the order of the figure

• include a no-insert negative control reaction, substituting dH₂O for insert DNA

Step 4

Incubate the reaction at room temp for 15 mins

Theory

Molar Ratio

• the ratio of the number of one type of molecule to another

  • in this case, the ratio of number of plasmid molecules to insert molecuels

• the ratio of plasmid:insert can affect ligation success, but the optimal ratio can’t be determined ahead of time, so a number of ligation reactions w/ variable ratios are set up

• since both molecules are DNA, are are relatively long, the molecular mass of each molecule is directly related to their length

  • in the formula, we can substitute molecular mass for bp

Molar Ratio Example Calculation

• here we’re setting up a 1:1 molar ratio, using 50 ng of cut plasmid

Ligase Enzyme

• natural function is to repair double-strand breaks in genomic DNA

• formation of two covalent bonds: phosphodiester bonds between 3’ OH of one nucleotide, with the 5’ phosphate end of another nucleotide

• reaction is ATP-dependent

Sticky Ends

• sticky ends can be more efficiently ligated than blunt ends

• where complementary overhangs are generated, the nucleotide polymers are held in place by H-bonds, which allows DNA ligase to function more efficiently in joining the strands

• most commonly used ligase is T4 ligase, derived from a bacteriophage. Works best at 25ºC but will function at lower which may promote the complementation of sticky DNA ends