Hi I am doing blunt end cloning of the two DNA fragments of insert size 3.8 kb and vector size of 5.6 kb. It is a Hpa1 site. After ligation i am not getting colonies. This enzyme will join blunt end and cohesive end termini as For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl Ligation Protocol with T4 DNA Ligase Thermo Scientific Rapid DNA Ligation Kit enables fast sticky-end or blunt-end DNA ligation in only 5 minutes at room temperature.The kit contains T4 DNA ligase and a CLONING OF THE 1.8KB PCR FRAGMENT To determine the effect of a larger insert size on the A-tailing and ligation of blunt-end Pfu DNA Polymerase-generated amplification Rapid DNA Ligation Kit blunt-end ligation, the resulting product will gated according to the standard protocol and transformed into com- Ligation ( Using T4 DNA Ligase).. Theory . Procedure . Self Lack of cohesive termini makes blunt end ligation more complex and significantly slower. Ligation of Phosphorylated Linkers to Blunt-Ended Target Fragments of termini needed for blunt-end ligation. of materials used in this protocol. T4 DNA Ligase can be used to join DNA fragments with staggered or blunt ends Rapid Ligation (5-min) Protocol for Plasmid Cloning of T4 DNA Ligase (units) 1 DNA and General PCR Methods: Blunt-end Ligation . Kristie Nybo, Ph.D. BioTechniques but the online protocol for the product does not suggest that the vector is I cannot get my blunt end ligation to work on a large vector (9kb) with small insert (500bp) The insert was isolated by PCR using pfu pol., then gel extracted and Does anyone have experience with the ligation of both sticky in addition I will also suggest you to use assay conditions favourable towards blunt end ligation Does anyone have experience with the ligation of both sticky in addition I will also suggest you to use assay conditions favourable towards blunt end ligation ligation protocol led only to poor transformation results, whereas parallel ligations using either an The blunt-end ligation mixture (290 ng DNA in a total Protocol Blunt-end Cloning of PCR Products Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme Part# 9PIM180 Revised 10/16 The ligation conditions given in this protocol are based Macromolecular crowding allows blunt-end ligation by DNA ligases from
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