Ligation+Protocol

Ligation Protocol 081511 __Reaction Contents__ Total Volume: 20 uL smaller volumes are actually better (5 or 10 uL reaction); in order to create such a small reaction it may be necessary to ethanol precipitate all of the the DNA reactants. The concentration of the DNA can be estimated by assuming that 90% of the DNA present before the ethanol precipitation is present.

DNA 2 uL 10X T4 DNA Ligase Buffer 1 uL T4 DNA Ligase -we use Promega T4 DNA Ligase 0.5 uL 70 mM ATP (not dATP!) y uL H20 Quick Ligase Incubation: Incubate at RT for 10 min Promega Ligase Incubation: Incubate 15 C for 16 hours then hold at 4 C in a thermocycler Optional heat inactivation step for some ligases (for example, 80 C 20 min)
 * Digested Fragment
 * Digested Plasmid
 * Shoot for 20-50 ng (for a library may want to use up to 200 ng DNA) (actually Kathy mentioned I should use 50 ng per 10 uL for a good ligation) total DNA in a manner such that there is a 3:1 molar ratio of insert to vector so that there is more insert than vector.
 * Spreadsheet for calculating ligation reaction: "C:\kurt\storage\CIM Research Folder\DR\2012\10-10-12\Ligation\Ligation Calculator.xlsx"
 * Note one might want to do an additional reaction with only plasmid and no fragment to measure the effect of plasmid self ligation.
 * last seen -20 #62 2nd shelf top left rack 2nd box back
 * Andrey suggests it is best to keep this ATP in small aliquots and don't use them more than 2 or 3 times. The ATP improves the ligation efficiency because the ATP in the buffer degrades after many freeze/thaw cycles over time.
 * last seen in 70 mM ATP Stock box in -80 C (#77 2nd shelf from bottom) (originally Andrey's box)

include component="page" page="Precipitation Protocol After Ligation" editable="1" wrap="1"

A Ligation Spreadsheet