Difference between revisions of "Part:BBa K349303"
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===Usage and Biology===
 
===Usage and Biology===
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This part is compatible with the Team Alberta 2010 Biobytes 2.0 assembly method. It can be utilized as a BA BioByte following digestion with BsaI and purification. The following protocol outlines the Biobytes 2.0 assembly method: <br>
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1) Mix the iron micro beads for 10 minutes.<br>
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2) Transfer 20 ul of iron micro beads to a 1.5 mL tube. <br>
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3) Pull the beads to the side using a magnetic tube rack. <br>
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4) Remove and discard the supernatant. <br>
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5) Add 50 ul of BioBytes 2.0 Wash Buffer to the beads. Flick gently to resuspend. <br>
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6) Pull the beads to the side using a magnetic tube rack. <br>
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7) Remove and discard the supernatant. <br>
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8) Repeat steps 5 to 7. <br>
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9) Add 200 ng of a premade anchor-Byte construct (premade in the GENOMIKON kit)  to the beads and top off the volume to 20ul with TE Buffer. Flick gently to resuspend. <br>
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10) Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube. <br>
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11) Repeat steps 6 and 7. <br>
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12) Repeat steps 5 to 7. <br>
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13) Add 200 ng of the next BioByte of your construct, making sure that a BA Byte follows an AB Byte and vice versa. Add an appropriate amount of 2x QuickLigase Buffer, Quick Ligase and TE to a total volume of 20ul. <br>
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14) Flick gently to resuspend.  Allow ligation for five minutes, flicking gently every minute. <br>
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15) Add 30ul of Wash Buffer to the tube. Flick gently. <br>
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16) Repeats steps 6 and 7. <br>
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17) Repeat steps 5 to 7 twice. <br>
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18) Repeat steps 13 to 17 for each subsequent Byte addition, including the last BioByte. <br>
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19) After the last Byte has been ligated and washed, add 30 ul of 75C Elution Buffer. Keep at 75C for 10 minutes. <br>
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20) After 10  minutes, still at 75C, put the tube into a magnetic rack . Allow the beads to be pulled aside and collect the supernatant into a clean 1.5 mL tube. The supernatant will contain your construct. <br>
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Revision as of 00:59, 28 October 2010

tetR-BA (BioBytes 2.0)

Tetracycline resistance cassette in the AB format of Biobytes 2.0.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 1300