Cloning a gene Flashcards
How do you do cloning by complementation?
Transforming a DNA library hopefully containing a WT copy of a mutant gene into a mutant cell, then scoring the transformants for a restored phenotype and minipreping the plasmid out of the transformant with the restored phenotype
If we don’t get a transformant with a restored phenotype from doing cloning by complementation, what are 3 possible reasons why?
- Library was incomplete and you didn’t have YFG+ in it
- The mutation was dominant
- There was a successful transformant but it wasn’t expressed
What is another name for cloning by complementation?
Plasmid rescue
What are 3 possible reasons for seeing a restored YFG phenotype, but there is no plasmid containing YFG?
- Overexpression of another gene suppressed the effects of the original mutation
- Transvection
- Original mutation reverted
What are 3 controls you would use for cloning by complementation?
Empty vector, plasmid curing, make a deletion based on the recovered clone
Why would you do empty vector transformation as a control for cloning by complementation?
Proves that the restored phenotype is actually because of the YFG sequence on the vector and not the backbone
Why would you do plasmid curing as a control for cloning by complementation?
If losing the plasmid causes the mutant phenotype again, we can say it was YFG+ that is restoring the phenotype
After doing cloning by complementation, how do you be extremely sure that it YFG is the mutant gene?
One step gene replacement. Proves thats the mutant gene if replacing YFG+ in the genome generates the same phenotype as the original mutation
What do you need to know about YFG to do one step gene replacement?
Upstream and downstream sequences in the genome. Get those by sequencing the vector
What is in the PCR template to do one step gene replacement?
A selectable marker that will replace YFG
What are the PCR primers for one step gene replacement?
3’ ends will bind to the template, and the 5’ ends have sequences that are homologous to regions upstream and downstream of YFG in the genome
How does one step gene replacement tell us for sure that we have the right gene cloned?
If we replace the suspect gene with something else and get the mutant phenotype, that shows it’s the gene
How can we get the original mutation once we know our gene?
Gapped plasmid repair
What is a gapped plasmid?
Most of the YFG sequences are cut out with a restriction enzyme, leaving a small region of homology
What happens if we transform a gapped plasmid into the original mutant?
The cell machinery will repair the gap to look like the original mutation in the genome, then we can miniprep that back out and sequence our original mutation