Lab 4 Flashcards
Describe plasmids.
Plasmids are extra-chromosomal DNA (“in addition” to the chromosome).
They are:
- Double –stranded
- Circular,
- usually supercoiled (compact, left),
- but when one strand is nicked, it becomes an “open” circle (right),
- replicate autonomously,
- much smaller than the chromosome,
- don’t contain “essential” genes, but genes that may confer an advantage in come environmental conditions or genes associated with bacterial pathogenesis.
What needs to happen to isolate a plasmid?
- lyse or break open the cell (i.e., destroy the membrane, peptidoglycan) without destroying the DNA
- Gram +ve cells – harder to lyse due to thick peptidoglycan
- Gram –ve cells – have outer (and inner) membrane
How is DNA released from bacterial cells in plasmid isolation procedures?
- With a lysis buffer containing:
- SDS - a detergent that helps to solubilize the lipid bilayers, and
- Lysozyme (and sometimes lysostaphin) - enzymes that degrade peptidoglycan
After DNA is released from the bacterial cells, how are plasmids isolated from large chromosomal DNA?
-
The alkaline lysis method - addition of NaOH after the cells have been suspended in the lysis buffer
- The addition of NaOH results in the chromosomal DNA and proteins being denatured, but the small plasmid DNA is usually not.
- Then the solution must be neutralized.
After addition of NaOH during plasmid isolation, the solution must be neutralized.
How is this done?
- By adding KOAc:
- Chromosomal DNA and proteins are denatured and precipitate out of the solution
- Small plasmid DNA does not denature and stays in solution
- Using a centrifuge, the precipitate (including chromsomal DNA and proteins) may be separated out from the solution, leaving the plasmid DNA soluble in the solution.
After removing the chromosomal DNA and proteins, how is the plasmid DNA precipitated out of the solution?
- By addition of ethanol
- Or, in a different kit:
- Instead of using ethanol, the solution is added to a column. The plasmid DNA binds to the beads in the column, And is then eluted from the beads with a small volume of buffer.
Plasmid DNA can be separated from chromosomal DNA on the basis of size and conformation (degree of supercoiling).
Describe the alkaline lysis method.
- DNA is denatured with sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS, a detergent) at alkaline pH (»pH 12.0).
- When potassium acetate (KOAc) is added to neutralize the NaOH, the denatured DNA strands attempt to re-anneal.
- The chromosomal DNA, due to its large size, cannot re-anneal and becomes trapped in the K/SDS complex and can be centrifuged out.
- The plasmid DNA is smaller and easily renatures upon addition of KOAc.
- The renatured plasmid remains soluble in the solution and can be found in the supernatant.
Describe the BioBasic Plasmid Miniprep kit.
- One example of a rapid procedure for the isolation of plasmid DNA using the alkaline lysis method and a silica-based membrane in a small spin column.
- All procedures are performed using micro-centrifuge tubes and micro-centrifuges.
- Following cell lysis and neutralization, the supernatant (containing plasmid DNA) is loaded into the spin column, which allows the DNA to adsorb onto the membrane.
- A wash solution is applied to remove contaminants from the column.
- The DNA is then eluted (released from the membrane in the column) by applying an elution buffer.
- Kits have an advantage is that they do not require any organic extractions (potential hazards) and can be accomplished in a short period (15 minutes or less for an experienced technician).
What are 2 advantages of using biobasic plasmid miniprep kits to isolate plasmid DNA?
Kits have an advantage is that they (1) do not require any organic extractions (potential hazards) and (2) can be accomplished in a short period (15 minutes or less for an experienced technician).
