InFusion-cloning (Fa. Clontech)

Materials

InFusion Kit Becton-Dickinson
Aqua bidest
Linearised vector (pDNR-Dual), 100ng/1µl (~4900bp)
Fragment (10-50ng/µl)
-> Subsequent method: Transformation

Procedure

• fragment concentration (fragment : pDNR-Dual  2:1 ratio [fmol])
• solution of a dry-down reaction in 102 µl aqua bidest
• add 14 µl 10x In-Fusion-buffer and 2 µl pDNR-Dual (200 ng)
• disperse to 15 tubes (8 µl per tube) and add 0.5 -1 µl of the fragment (~ 2:1 ratio with regard to vector)
• fill up to 10 µl with aqua bidest and incubate for 30 min at room temperature
• put tubes on ice to stop the reaction
• transformation in E.coli nova blue and plating on LB-amp100