Difference: ProtocolsAraMarker (12 vs. 13)

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The Arabinose (Ara) Genetic Marker

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  1. At this point, you should have several colonies on most plates. They might appear as small colonies after only 24 hrs of growth, but it's safer to wait two days before proceeding. Sometimes a plate will have zero cells. Sometimes it might have hundreds or thousands (a jackpot). For more about this distribution, read about fluctuation tests.
  2. Pick ONE colony from each plate and streak it out on a new MA plate. This extra step ensures that you have isolated your new strain from the hazy background of Ara– cells and that it is not a mixture of two Ara+ revertants. Store the old plates at 4°C until you are sure your new streaks have grown.
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  1. Incubate new MA plates at 37°C for 36–48 hours.
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  1. Incubate new MA plates at 37°C for 24–48 hours.
 

Day 6:

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  To verify that you have the precise reversion present in REL607 in your new Ara+ revertant candidate, you can use PCR followed by digestion with a restriction enzyme that cuts only when the REL607 allele is present.
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Perform a standard PCR reaction from whole cells using these two primers. *Always include REL606 (Ara+) and REL607 (Ara–) controls when doing this assay. You can use cells from a freezer stock.
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Perform a standard PCR reaction from whole cells using these two primers. Always include REL606 (Ara+) and REL607 (Ara–) controls when doing this assay. You can use cells from a freezer stock.
 
Primer Coordinates Sequence
REL256 REL606/70660-70683 5'-CCGATACGCTCATGGGCTTGTTTA-3'
 
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