Blotting and sequencing exam III.txt
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Southern & North blotting steps
- SOuthern blot: looks for specific genes (DNA) in sample
- Northern Blot: looks for RNA (not DNA)
- 1. run on agarose gel
- 2. soak gel in base (NaOH): denatures dsDNA --> ssDNA
- 3. Transfer DNA to a blot (membrane, usually nitrocellulose) and let sit overnight
- 4. Prepare a PROBE and probe the membrane: probe will be complementary to the gene looking for. probe can be labelled w/radioactivity (ie p32)
- 5. Roll membrane inside tube & insert probe
- 6. Remove blot and wash off excess label (p32)
- 7. Expose membrane to film
- 8. Develop film to see print on film of blot
Blotting technique w/capillary action
- let sit overnight in the following apparatus:
- (top to bottom)
- weight (book)
- paper towels
- buffer solution underneath
- used to identify a specific protein
- once gel transferred to blot, the blot is soaked in milk to bind other spots where protein isn't present (bc milk has a lot of proteins)
- 1ary antibody: make antibody against protein, which binds to the specific protein
- 2nd antibody: Ab which binds to 1ary antibody, which forms a Precipitate when bound to 1ary antibody
- Uses rxn groups (PCR mixes) which incorporate ddA, ddT, ddG, ddC. and during PCR the sequence.
- The primer which is complement to template strand will add on the rest of the sequence, and each mix will make different segments, showing how many bps it is until you get to an A, for example.
- You can order the sequence when looking at the small to large bands on the gel u run.
- the COMPLEMENT of this sequence is what you are looking for!!
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