Berglund:Cloning: Plasmid ligation

Materials
Cut plasmid Cut PCR product (same cuts as the plasmid) T4 ligase and T4 ligase buffer (commmercially prepared) 5XKCM 1.25ml of 4M KCL (fc=0.5M KCl) 1.5ml of 1M CaCl2 (fc=0.15M CaCl2) 2.5ml of 1M MgCl2 (fc=0.25M MgCl2) 4.75 ml nanopure water

Methods
Ligation 4.5 µl	water 2 µl	cut PCR (cleaned and in water) 2 µl	cut plasmid (cleaned and in water) 1µl	T4 ligation buffer 0.5µl	T4 ligase Total volume=10µl Ligate for 1-3 hrs at room temperature or for overnight at 4-16C in the fridge. Transformation 1µl-2µl of ligated plasmid 59µl of water 40µl of 5x KCM 100µl of competent cells Berglund:Making_Competent_Cells:_Various_Methods
 * 1) incubate on ice for 20 min
 * 2) heat shock for 90 sec at 42°C
 * 3) add 80µl SOC
 * 4) plate all of it on LB-amp