Vector 1 (Insert 1, Insert 2, Insert a ) and Vector 2 ( Insert 1, Insert 2, Insert b) contain three inserts in which only the Insert a and b are different. These two vectors are constructed by Gibson assembly and transformed into E.coli DH5A cells. Positive clones are obtained based on antibiotic resistance. Plasmid was isolated for further cloning. While PCR check, Insert 1 &2 of vector 1 are amplified and visualised as a bright band, whereas when the same primers were used for amplification of Insert 1 and 2 in vector 1, it is lighter. The template concentration of vector 1 was also varied and checked.
Apart from primers specific to inserts 1 and 2, primer was designed for upstream and downstream regions of inserts. In that case, I am getting proper amplification for both vectors.
What could be the reason for getting different intense bands when the same primers are used for the amplification of the same inserts in different vectors?
Have you factored in differences in the size of the vectors? If one is bigger than the other you will be loading your PCR reaction with a lower copy number which will affect the band intensity.
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