Plasmid DNA purification steps - 1. grow the bacteria with the plasmid
2. centrifuge
3. remove supernatant
4. resuspend pellet in lysis buffer (detergents and high salt)
5. neutralize the lysis buffer
6. spin down to remove debris and genomic DNA
7. put the suspension on a spin column
8. plasmid DNA will adhere to spin column and everything else will be washed away
with high salt buffers
9. add low salt buffer to remove plasmid DNA from spin column
RNA purification steps - Same steps as DNA purification, but also need to heat to
disrupt any secondary structures.
Since the mRNA is the only type that has a polyA tail, the beads can have a "polyT tail"
(oligo dT) that will hybridize to the mRNA and keep it in the column.
Then wash to remove everything that is not mRNA.
Wash with high salt buffer to release the mRNA.
https://www.youtube.com/watch?v=yzqhPBAvvY0
Gel Electrophoresis - Used to separate DNA fragments of different sizes.
1. prepare gel
2. load DNA
3. run gel
4. visualize
What direction does the DNA move during gel electrophoresis? Why? - DNA moves
toward cathode (+) because it is negatively charged (PO4- backbone)
Agarose gel - Used for gel electrophoresis because it has small pores that allow for
DNA movement
For large fragments of DNA, do you want a high or low agarose concentration? - Low.
This will allow the large fragments to move farther apart and it will be easier to visualize
them (.7%)
, For small fragments of DNA, do you want a high or low agarose concentration? - High.
This will allow for better visualization of the small fragments. (1.5%)
DNA staining - Ethinium bromide is a carcinogen because it intercalates into the DNA.
Karyotyping - Allows for chromosomal visualization.
What are the bands in karyotyping? - The light bands are GC rich areas and the dark
bands are AT rich areas.
What can karyotyping detect? - Large chromosomal deletions, insertions, and
translocations. Can diagnose diseases like Down Syndrome (extra 21st chromosome)
or Turner's Syndrome (missing an X chromosome)
Microdissection of human chromosome - Stop the cell cycle during M phase (when DNA
is most condense) and then break the nucleus and locate band of interest
FISH - Used for detection of chromosomal rearrangements. Can detect deletion,
amplification, translocation
Can FISH only be used with DNA - No - can also be used with RNA to study gene
expression (mRNA)
FISH steps - 1. make probe ssDNA (complementary with DNA of interest) with
fluorescence tags
2. denature probe and target DNA
3. wash target with probes and allow to hybridize
4. visualize
FISH deletion diagnosis - PTEN (tumor supressor) can be deleted and FISH can detect
this. Label the target DNA with probes for the PTEN gene and visualize whether there
are genes in the chromosomes
FISH amplification diagnosis - C-MYC (type of leukemia) can be diagnosed with FISH.
FISH translocation diagnosis - CML (type of leukemia) can be diagnosed with FISH.
results from fusion of two genes. Mark those two genes and (red and green) and look
for any yellow overlap in target DNA. Gleevec is a blocker for this fusion
DNA sequencing - Sanger Sequencing - 1. break DNA into smaller fragments
2. anneal primer
3. carry out 4 rxns (one with each halting neucleotide)
4. run gel
5. you can then figure out the sequence of the complimentary strand
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