Term
| What do restriction endonucleases do? |
|
Definition
| They cut DNA at specific places |
|
|
Term
| What is special about the sequences that restriction endonucleases cut? |
|
Definition
|
|
Term
| What is on the ends of DNA after they are cut? |
|
Definition
|
|
Term
| What is a famous restriction enzyme? |
|
Definition
|
|
Term
| Are restriction enzymes specific? why/why not? |
|
Definition
| They recognize specific sequences that are 4,6, or 8 nucleotides long, so that's really hard to have like a random cut somewhere. |
|
|
Term
| What does the term "sticky" ends mean in reference to some restriction enzymes? |
|
Definition
| They can leave one strand longer than the other. BUT, not all restriction enzymes do this. |
|
|
Term
| What do restriction endonucleases do for bacteria? |
|
Definition
| They cut up viral DNA--primitive immune system. |
|
|
Term
| Why does bacterial DNA not get chopped up by their restriction enzymes? |
|
Definition
| The sites where the endonucleases would cut are heavily methylated to protect them. |
|
|
Term
| What is a plasmid cloning vector? |
|
Definition
| You use a plasmid plus an endonuclease to cut DNA, insert the DNA into the plasmid, and then the plasmid will replicate itself because it has an ORI, so you can clone that DNA sequence alot. |
|
|
Term
| How can you tell if the bacteria are taking up the sequence in plasmid cloning? |
|
Definition
| You can add an antibiotic resistance gene in there too, so if you add antibiotics, only the bacteria that got your sequence will live. |
|
|
Term
| What is the process of using a plasmid cloning vector? |
|
Definition
| Cut the plasmid with EcoRI to expose the sticky ends. Add the chromosomal DNA which has been cut by EcoRI. The complimentary ends will stick together. Add DNA ligase to join together the ribonucleotides. |
|
|
Term
| Besides the antibiotic method, what's another way to verify that you put the gene into the plasmid? |
|
Definition
| Get a pure solution of plasmids. Cut them again with EcoRI. Analyze with gel electrophoresis. |
|
|
Term
| If you cut a plasmid cloning vector with the restriction endonuclease and run a gel, how many bands will it have? |
|
Definition
| Two bands. One for the vector and one for the fragment. |
|
|
