I am doing Pcr cloning trying to clone a 3000 bp pcr product into a 9000 bp pcdh_bec1 plasmid .digestion was done using Xba1 and Not1 ,after gel extraction loaded 2ul on the gel and observed faint band of backbone. for ligation used 2ul(10ng/ul) backbone and 17ul of the insert(24ng/ul). didn't get any colonies I am using stbl2 cells growing them at 30 degrees to reduce chances of recombination.
when i followed the same strategy with the Plvx vector using the same competent cells i observed good amount of colonies although efficiency of competent cells was low.
Can someone help?
Ligation efficiency might be low. Considering your competent cells are also low in competency, the probability of getting clones is further reducing. Increase ligation efficiency by increasing backbone and insert DNA amounts or try ligation at different temperatures, or increase competency of cells. We use Top10 cells which are highly competent, you can try similar type of cells
Hello. It is better to use the TA cloning method for cloning large pieces. You can also make changes in your ligation to increase the efficiency of the reaction. You can use the site https://nebiocalculator.neb.com/#!/ligation to get the exact values of the vector and insert, in the ligation reaction. Also, changing the strains of bacteria used in transformation or their preparation method can help you. TOP10 bacteria is a good option for preparing competent bacteria using calcium chloride.
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