26 February 2021 7 3K Report

I am facing difficulties in cloning a 1kb gene into a vector (pJIT163). I have my gene on interest (GOI) in pUC57 and want to clone in pJIT163 using SalI and BamHI restriction sites. I am getting a lot of colonies like thosands colonies/plate on each plate including +ive, -ive and test plates. I am using LB-agar media containing 50 ug/ul of ampicillin as selection marker. My plates were designed as +ive = pJIT163, -ive = competent cells (cc), L1-3 = ligation reaction performed on different days.

-*Double digestion of pUC57 and pJIT163 with SalI + BamHI

-Got expected bands

-Gel purification

-*Ligation at 18-22 degree overnight

*No heat inactivation

Next transformation

-10 minutes thaw at 4 degree

-4ul ligation rxn per 20 ul cc

-35 minutes on ice

-45 seconds of heat shock at 42 degree

-2-5 mins on ice

-added 100 ul LB media

-1 hour of shaking incubation at 37 degree

-Spreading over LB-agar plate containing 50 ug/ul of ampicillin as selection marker

-16-17 hours of incubation at 37 degree

Can anyone suggest why am I getting colonies over -ive, +ive and test plates as well? First, I suspected ampicillin solution is hydrolized or out dated; I changed the amipicllin solution and now same results.

Now, I am suspecting my cc got ampicillin resistant by any means. Any other suggestions.

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