Lecture 6 Restriction Digests Flashcards
What determines migration rate on DNA gel electrophoresis?
Dependent on molecular size (for linear fragment), conformation (circular, nicked circular or supercoiled DNA do not behave the same as linear DNA)
What are the diff. types of stains for gel electrophoresis?
When exposed to UV light, ethidium bromide/DNA Safe Stain will fluoresce:
EtBr gives a reddish-orange colour which intensifies almost 20-fold after binding to DNA
DNA Safe Stain usually gives green or red depending on the kind of molecule used for fluorescence (SYBR Green or Gel/Red)
What is the role of gel percentage?
To visualize DNA separation at different gel %
Larger fragments separate better at lower %
Smaller fragments separate better at larger %
How can you differentiate Plasmid and Genomic DNA on a gel?
Plasmid DNA is shaped of a nicked circle
Genomic DNA is a smear on gel
- degrading hundreds of small and big fragments
- continuous progression from small to large
If RNA is not digested or cut then it is supercoiled/blob
How to quantify DNA on a gel?
The brighter the fluorescence, the more DNA is on the gel
Concentration of linear DNA can be determined if known standards are loaded on the gel (compare to standards)
RNA will be at the bottom of the gel
What is the diff. between complete and partial digest?
Complete
- Every restriction site is cut out
Partial
- Various fragments
What are reasons that can lead to partial digest?
- Time of incubation
- Amt of DNA added (too much)
- Temperature
- Not enough cation
- Too much salt and ions in DNA
- Not properly mixed DNA in epi tube
How can you determine the size of DNA fragments?
Determine MW of unknown DNA molecule using distance migrated by fragments of a known size
Migration is inversely related to the log MW (Mr)
Measure the distance migrated for each fragment and then line it up with the ladder with the known sizes and then plot
What are things to consider when setting up a restriction digest?
- Amount of DNA to be digested
- Enzyme(s) used
- Enzyme buffer requirements (final concentration 1X unless specified)
- Units of enzyme required (based on amt of DNA)
- Final conc. of glycerol must be 5% or less (Stock is shipped in 50% glycerol)
- Amt of water required if necessary
What is DNA ligation?
DNA ligase joins two fragments of DNA together and requires a phosphate group
Blunt vs cohesive ends (cohesive is more efficient)
Ligation of Vector and Insert
Must cut vector with appropriate restriction enzymes
Ligation of a segment of foreign DNA into a linearized plasmid vector involves the formation of new bonds between 5’ phosphate and adj. 3’ - hydroxyl (forms a phosphodiester bond)
