Hello everyone,

I am currently working on a cloning project with 2 vector pBin mCherry (for localization) and pG5H-GFP (for gene expression). I need to clone 1 ATG gene with the size of 1.3 kb to both vector. For pBin mCherry, the RE I chose is Xbal I and Sal I, as for pG5H-GFP both end are attached with MIu I.

At first, I cloned the ATG to pGem T easy vector and digested with selected enzyme ( I attached imgae as below) then I digested the vectors with the same RE set (image as below). pG5H-GFP even before be digested by RE, it already has 2 bands and both of them need to be included for successful ligation, so after digestion, I couldnot do gel extraction like other vectors.

-When calculated for the amount of input enzyme, I used this formula:

For 2.5ug of DNA we need 50ul of total volume and for 10ul of total volum we need 0.2ul of RE.

Because pG5H-GFP has same RE at both size, after digestion, I also performed CIAP clean up as following:

- for 0.165 pmol DNA we need 0.5ul of 10x CIAP in 50ul total volum

- the pmol of DNA is calculated = M (conc of DNA in ug) x 1514/size of vector (bp)

However, after transformation, I still got alot of clony on self-ligated pG5H - GFP. I also got alot on ATG ligated plate, but after colony PCR, they appeared to be vector only.

And I could not obtain any colony on pBin plate (both self-ligated and gene ligation).

I keep getting this problem with this gene so I hope everyone could help me to solve it.

Thank you so much.