9,292
edits
BISC219/F12: RNAi Lab 7: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
→Picking a Bacterial Colony
Tucker Crum (talk | contribs) |
Tucker Crum (talk | contribs) |
||
| Line 44: | Line 44: | ||
== Picking a Bacterial Colony == | == Picking a Bacterial Colony == | ||
Your instructor will give each pair an LB+ampicillin plate on which bacteria containing our pPD129.36 ''lsy-2'' plasmid are selectively growing. Please pick two well isolated colonies, circle them, label one A and the other B and write your initials and group color on the agar side of the plate. <br><br> | Your instructor will give each pair an LB+ampicillin plate on which bacteria containing our pPD129.36 ''lsy-2'' plasmid are selectively growing. Please pick two well isolated colonies, circle them, label one A and the other B and write your initials and group color on the plastic bottom (agar side) of the plate. <br><br> | ||
How can you be sure that your colonies contain the plasmid carrying the gene you are interested in studying? In theory, any colony of bacteria growing on your LB+amp plate should contain a plasmid because the gene for antibiotic resistance is not chromosomal, but instead expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. The amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme; therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. You must be careful to pick ONLY the bigger, central colony and not the satellites. The satellite bacteria are unlikely to carry the plasmid that contains the ''C. elegans lsy-2'' gene.<BR><BR> | How can you be sure that your colonies contain the plasmid carrying the gene you are interested in studying? In theory, any colony of bacteria growing on your LB+amp plate should contain a plasmid because the gene for antibiotic resistance is not chromosomal, but instead expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. The amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme; therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. You must be careful to pick ONLY the bigger, central colony and not the satellites. The satellite bacteria are unlikely to carry the plasmid that contains the ''C. elegans lsy-2'' gene.<BR><BR> | ||
