Molecular Techniques
Background Information
Notes on Using Gibson Cloning to swap genes/domains between two plasmid containing clones: The best way move part/all of gene one in plasmid 1 to gene 2 in plasmid 2 is to amplify your target vector with primers that create ~25 base pair overlaps to the domain you'd like to swap in. The product of this PCR should be a linear fragment that ends at the start and end of the gene fragment you're going to be adding in, minus the current fragment you will be replacing. Then you can digest this amplicon with DpnI to remove background vector, clean up, and assemble with the gene fragment you want to swap in.
Notes on NEB SDM: If time permits, run gel on PCR reaction to identify non-specific bands. Also if primers have different Ta for SDM thermocycle, you can run those with Ta’s within 5oC of each other. Use the lower Ta. To avoid primers with different Ta’s, selecting the option to confine the change to the 5' tails, this would allow you to better make adjustments to the length of the resulting primers (by hand) if needed. The NEB Tm Calculator could be more readily used to check the Tm if any adjustments are needed if the change is on the tail. You could also potentially set a different minimum Tm for the primers, if you are finding some sets are quite a bit lower than others, again when confining the mutation to the 5' tail using basecbanger.
Protocols
Cloning by PCR AddGene / NEB
Tutorials/ Videos
PCR Cloning Tutorial NEB
Teaching Files
Molecular Biology Basics Exam
Handout on Bacterial Culture
Handout on DNA Cloning and SDM
Something missing or do you have something to add? Email Josephprovost@sandiego.edu
