Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Cloning fragments with blunt ends - (Nov/22/2006 )


I will make a digestion with ScaI enzyme of BAC that it is contains an interest locus, for later cloning the fragments obtained in pCR-XL-TOPO (Invitrogen).
In theory I can use the EcoRV enzyme for the digestion of pCR-XL-TOPO, after digesting do I Have to proceed to desphosphorylation (CIAP), ligation and transformation finally in cells TOP10 (Competent Cells). I really say thanks yours opinions, tips and experimental steps (according to yours experiences).



do you mean whether you have to dephosphorylate your blunt-end plasmid or not? huh.gif

if so, i think blunt ends have less opportunity to self-ligation even if they 're not dephosphorylated....but i think it's recommended to dephosphorylate anyway unsure.gif


dephospho for blunt ends is improved as promega mentionned by doing
1µl CIP
15' 37°
15' 56°
re add 1µl CIP
15' 37°
15' 56°
lso ligation was helped by adding 10% PEG 6000 (colleague personnal experience)