09 October 2024 2 3K Report

I am cloning a gene (903bp) into miniTurbo_NLS (6338 bp).

  • I PCR amplified the insert from it's backbone to introduce EcoR1 and Xho1 sites. It was run on gel and eluted. I then double digested it for 5 hrs at 37oC and was eluted from gel.
  • I double digested the vector for 5 hrs at 37oC and did alkaline phosphatase treatment and inactivated. I then ran it on gel giving a band of 6248 bp and eluted. Note- I didn't confirm the 90 bp fallout.
  • The vector and insert was ligated in 1:3 ratio but no colonies grew after transformation into TG1 chemically comp cells.
  • Ligation was attempted again with 1:7 ratio and only 1 colony grew.
  • The control plate with vector only + ligase had 3 colonies.
  • Another control plate with vector only - ligase had no growth.
  • The single colony that grew was insert specific colony pcr negative.
  • Even after isolating the plasmid it was negative.

How to make this cloning work ?

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