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The Arabinose (Ara) Genetic Marker | ||||||||
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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. | ||||||||
Changed: | ||||||||
< < | 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. | |||||||
> > | 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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The Arabinose (Ara) Genetic Marker | ||||||||
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< < | Selection for arabinose utilization from REL606 is known to also produce, more rarely, Ara+ revertants that have an araA 92A (GCC) sequence. | |||||||
> > | Selection for arabinose utilization from REL606 is known to also produce, more rarely, neutral Ara+ revertants that have an araA 92A (GCC) sequence. | |||||||
Selection of Ara+ Revertants from Ara− Strains Derived from REL606 |
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The Arabinose (Ara) Genetic Marker | ||||||||
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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. | ||||||||
Changed: | ||||||||
< < | 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. | |||||||
> > | 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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The Arabinose (Ara) Genetic Marker | ||||||||||
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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. | ||||||||||
Changed: | ||||||||||
< < | Perform a standard PCR reaction from whole cells with these two primers. | |||||||||
> > | 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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< < | Cut with HaeII and run on a 3% agarose gel. Ara+ with the REL607 reversion mutation will show a shift of the 226 bp band down into the composite ~200 bp band because this mutation creates an additional copy of the restriction site. | |||||||||
> > | Use a 1 minute extension time. This PCR produces a 495 bp product. Next, cut the PCR product with the restriction enzyme HaeII. It is not necessary to use a PCR cleanup kit before this step. To do this, make a master mix consisting of 3.8 µl dH2O, 1 µl 10x NEBuffer 4, 0.1 µl 100x BSA (10 mg/ml), and 0.1 µl of HaeII (20,000 U/ml). Add 5 µl of this master mix to 5 µl of PCR sample. The amount of enzyme per reaction comes out to 2 U. Incubate samples in a water bath or PCR machine block at 37°C for >1 hr. Run the products on a 3% agarose gel. Load all 10 µl of each sample (adding an appropriate amount of load buffer to them). You will need good resolution of small fragments to see the results. Ara+ with the REL607 reversion mutation will show a shift of the 226 bp band down into the composite ~200 bp band because this mutation creates an additional copy of the restriction site. | |||||||||
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The Arabinose (Ara) Genetic Marker | |||||||||||
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General Procedure | |||||||||||
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< < | For a non-mutator strain. Grow several 10 ml cultures in DM1000 media (LB is also fine in most situations). Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara+ revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it is Ara+ and a clone. Then, grow an overnight culture in LB or DM1000 and store frozen. For a mutator strain, you can plate 1/10 to 1/100th as many cells and expect to get the same number of Ara+ revertants. | ||||||||||
> > | For a non-mutator strain. Grow several 10 ml cultures in DM1000 media (LB is also fine in most situations). Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara+ revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it is Ara+ and a clone. Then, grow an overnight culture in DM1000 (or LB( and store frozen. For a mutator strain, you can plate 1/10 to 1/100th as many cells and expect to get the same number of Ara+ revertant candidates. | ||||||||||
Detailed ProcedureSupplies | |||||||||||
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Day 1: Revive
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Day 2: Grow replicates | |||||||||||
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Day 3: Plate on Selective Media
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PCR-RFLP Assay for Verifying the REL607 Mutation |
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The Arabinose (Ara) Genetic Marker | ||||||||
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Selection of Ara+ Revertants from Ara− Strains Derived from REL606 | ||||||||
Changed: | ||||||||
< < | For a non-mutator strain. Grow several 10 ml cultures in LB or DM1000 media. Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara+ revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it is Ara+ and a clone. Then, grow an overnight culture in LB or DM1000 and store frozen. | |||||||
> > | General ProcedureFor a non-mutator strain. Grow several 10 ml cultures in DM1000 media (LB is also fine in most situations). Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara+ revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it is Ara+ and a clone. Then, grow an overnight culture in LB or DM1000 and store frozen. For a mutator strain, you can plate 1/10 to 1/100th as many cells and expect to get the same number of Ara+ revertants.Detailed ProcedureSuppliesDay 1: Revive
Day 2: Grow replicatesDay 3: Plate on Selective Media
Day 5:
Day 6:
Day 7:
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PCR-RFLP Assay for Verifying the REL607 Mutation |
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The Arabinose (Ara) Genetic MarkerBackground | ||||||||
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< < | The Escherichia coli B strain REL606 has a mutation in the araA gene that renders it unable to utilize the sugar L-arabinose. Strain REL607 is a spontaneous revertant of REL606 containing a single point mutation that restores the ability to metabolize L-arabinose. This marker is selectively neutral in a variety of conditions and can be used to determine the relative frequencies of Ara- (REL606-derived) and Ara+ (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara- and Ara+ cells form red and white colonies, respectively, on tetrazolium arabinose (TA) plates, because utilization of the sugar rather than only the tryptone and yeast extract components of this medium causes the excretion of acetic acid which acidifies the area surrounding the colony, changing the tetrazolium indicator color from red to white. | |||||||
> > | The Escherichia coli B strain REL606 has a mutation in the araA gene that renders it unable to utilize the sugar L-arabinose. Strain REL607 is a spontaneous revertant of REL606 containing a single point mutation that restores the ability to metabolize L-arabinose. This marker is selectively neutral in a variety of conditions and can be used to determine the relative frequencies of Ara− (REL606-derived) and Ara+ (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara− and Ara+ cells form red and white colonies, respectively, on tetrazolium arabinose (TA) plates, because utilization of the sugar rather than only the tryptone and yeast extract components of this medium causes the excretion of acetic acid which acidifies the area surrounding the colony, changing the tetrazolium indicator color from red to white. | |||||||
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< < |
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> > |
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< < | Selections for arabinose utilization from REL606 is known to also produce Ara+ revertants that have an araA 92A (GCC) sequence. | |||||||
> > | Selection for arabinose utilization from REL606 is known to also produce, more rarely, Ara+ revertants that have an araA 92A (GCC) sequence. | |||||||
Changed: | ||||||||
< < | Selection of Ara+ Revertants from Ara- Strains Derived from REL606 | |||||||
> > | Selection of Ara+ Revertants from Ara− Strains Derived from REL606 | |||||||
For a non-mutator strain. Grow several 10 ml cultures in LB or DM1000 media. Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara+ revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it isAra+ and a clone. Then, grow an overnight culture in LB or DM1000 and store frozen. | ||||||||
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< < | Cut with HaeII and run on a 3% agarose gel. Ara+ with the REL607 reversion mutation will show a shift of the 226 bp band down into the composite ~200 bp band because this mutation recreates a restriction site. | |||||||
> > | Cut with HaeII and run on a 3% agarose gel. Ara+ with the REL607 reversion mutation will show a shift of the 226 bp band down into the composite ~200 bp band because this mutation creates an additional copy of the restriction site. | |||||||
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> > |
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The expected result is that a majority (>50%) of selected Ara+ revertants have this exact mutation. |
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The Arabinose (Ara) Genetic Marker | ||||||||
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Selection of Ara+ Revertants from Ara- Strains Derived from REL606 | ||||||||
Changed: | ||||||||
< < | For a non-mutator strain. Grow a 10 ml culture in LB or DM1000 media. Spin down to concentrate cells and plate them all on a minimal arabinose (MA) plate. Grow for 48 hours, pick an Ara+ colony. Streak on an MA plate to verify and purify clone. Grow an overnight culture in LB or DM1000 and freeze down. | |||||||
> > | For a non-mutator strain. Grow several 10 ml cultures in LB or DM1000 media. Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara+ revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it isAra+ and a clone. Then, grow an overnight culture in LB or DM1000 and store frozen. | |||||||
Changed: | ||||||||
< < | RFLP Assay for Verifying the REL607 Mutation | |||||||
> > | PCR-RFLP Assay for Verifying the REL607 MutationTo 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. | |||||||
Perform a standard PCR reaction from whole cells with these two primers. | ||||||||
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> > | The expected result is that a majority (>50%) of selected Ara+ revertants have this exact mutation. | |||||||
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< < | Selecting Arabinose Marker Revertants | |||||||||
> > | The Arabinose (Ara) Genetic Marker | |||||||||
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< < | Background | |||||||||
> > | Background | |||||||||
The Escherichia coli B strain REL606 has a mutation in the araA gene that renders it unable to utilize the sugar L-arabinose. Strain REL607 is a spontaneous revertant of REL606 containing a single point mutation that restores the ability to metabolize L-arabinose. This marker is selectively neutral in a variety of conditions and can be used to determine the relative frequencies of Ara- (REL606-derived) and Ara+ (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara- and Ara+ cells form red and white colonies, respectively, on tetrazolium arabinose (TA) plates, because utilization of the sugar rather than only the tryptone and yeast extract components of this medium causes the excretion of acetic acid which acidifies the area surrounding the colony, changing the tetrazolium indicator color from red to white. | ||||||||||
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Selections for arabinose utilization from REL606 is known to also produce Ara+ revertants that have an araA 92A (GCC) sequence. | ||||||||||
Changed: | ||||||||||
< < | Selection of Ara+ Revertants from Ara- Strains Derived from REL606 | |||||||||
> > | Selection of Ara+ Revertants from Ara- Strains Derived from REL606 | |||||||||
For a non-mutator strain. Grow a 10 ml culture in LB or DM1000 media. Spin down to concentrate cells and plate them all on a minimal arabinose (MA) plate. Grow for 48 hours, pick an Ara+ colony. Streak on an MA plate to verify and purify clone. Grow an overnight culture in LB or DM1000 and freeze down. | ||||||||||
Changed: | ||||||||||
< < | RFLP Assay for Verifying the REL607 Mutation | |||||||||
> > | RFLP Assay for Verifying the REL607 Mutation | |||||||||
Perform a standard PCR reaction from whole cells with these two primers. | ||||||||||
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Selecting Arabinose Marker Revertants | ||||||||
Line: 14 to 14 | ||||||||
Selection of Ara+ Revertants from Ara- Strains Derived from REL606 | ||||||||
Changed: | ||||||||
< < | For a non-mutator strain. Grow a 10 ml culture in LB or DM1000 media. Spin down to concentrate cells and plate them all on a minimal arabinose (MA) plate. Grow for 48 hours, pick an Ara+ colony. Streak on an MA plate to verify and purify clone. Grow an overnight culture and freeze down. | |||||||
> > | For a non-mutator strain. Grow a 10 ml culture in LB or DM1000 media. Spin down to concentrate cells and plate them all on a minimal arabinose (MA) plate. Grow for 48 hours, pick an Ara+ colony. Streak on an MA plate to verify and purify clone. Grow an overnight culture in LB or DM1000 and freeze down. | |||||||
RFLP Assay for Verifying the REL607 MutationPerform a standard PCR reaction from whole cells with these two primers. | ||||||||
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< < | Primers | |||||||
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Selecting Arabinose Marker Revertants | ||||||||
Line: 14 to 14 | ||||||||
Selection of Ara+ Revertants from Ara- Strains Derived from REL606 | ||||||||
Added: | ||||||||
> > | For a non-mutator strain. Grow a 10 ml culture in LB or DM1000 media. Spin down to concentrate cells and plate them all on a minimal arabinose (MA) plate. Grow for 48 hours, pick an Ara+ colony. Streak on an MA plate to verify and purify clone. Grow an overnight culture and freeze down. | |||||||
RFLP Assay for Verifying the REL607 Mutation | ||||||||
Added: | ||||||||
> > | Perform a standard PCR reaction from whole cells with these two primers. | |||||||
Primers | ||||||||
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< < |
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Selecting Arabinose Marker Revertants | ||||||||||||||||
Line: 6 to 6 | ||||||||||||||||
The Escherichia coli B strain REL606 has a mutation in the araA gene that renders it unable to utilize the sugar L-arabinose. Strain REL607 is a spontaneous revertant of REL606 containing a single point mutation that restores the ability to metabolize L-arabinose. This marker is selectively neutral in a variety of conditions and can be used to determine the relative frequencies of Ara- (REL606-derived) and Ara+ (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara- and Ara+ cells form red and white colonies, respectively, on tetrazolium arabinose (TA) plates, because utilization of the sugar rather than only the tryptone and yeast extract components of this medium causes the excretion of acetic acid which acidifies the area surrounding the colony, changing the tetrazolium indicator color from red to white. | ||||||||||||||||
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> > |
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Selection of Ara+ Revertants from Ara- Strains Derived from REL606RFLP Assay for Verifying the REL607 MutationPrimers \ No newline at end of file | ||||||||||||||||
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Selecting Arabinose Marker RevertantsBackgroundThe Escherichia coli B strain REL606 has a mutation in the araA gene that renders it unable to utilize the sugar L-arabinose. Strain REL607 is a spontaneous revertant of REL606 containing a single point mutation that restores the ability to metabolize L-arabinose. This marker is selectively neutral in a variety of conditions and can be used to determine the relative frequencies of Ara- (REL606-derived) and Ara+ (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara- and Ara+ cells form red and white colonies, respectively, on tetrazolium arabinose (TA) plates, because utilization of the sugar rather than only the tryptone and yeast extract components of this medium causes the excretion of acetic acid which acidifies the area surrounding the colony, changing the tetrazolium indicator color from red to white.Selection of Ara+ Revertants from Ara- Strains Derived from REL606RFLP Assay for Verifying the REL607 MutationPrimers |