User:Morgan L. Paull/Notebook/Genetic Fence/2011/01/19

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Checking Genetic Fence Plasmids

  • Running digests to confirm the status of biobrick plasmids from igem team fence over the summer and into the fall. If the proper-sized cutout appears, I will keep the plasmid checked and discard the others so as to consolidate the materials in the fence boxes down to one or two boxes.
  • digested each plasmid with EcoR1 and Pst1 to confirm the presence of the widest parts of the biobrick sites as well.
  • used between 1 and 3μL of plasmid, depending on concentration, in an attempt to stay at reasonably concentrations of DNA.


First (top) Row

Number: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Name: KB+ Ladder Barstar 1 Barstar 2 RXRHm 2 RXRHm 1 LacIN 2 Gal4 DBD 1 Gal4 DBD 2 VP16GalEcR 1 VP16GalEcR 2 GalEcR 1 GalEcR 2 LacIN 1 5xGalUAS 1 5xGalUAS 2 LacIN Nost Act2LacO 1 LacIN Nost Act2LacO 2 KB+ Ladder
Expected Length(bp): 273 273 600+ 600+ 1119 438 438 714 714 1446 1446 1119 93 93 2657 2657
Correct Size: yes + yes + yes + yes + yes + yes yes + yes yes short yes blank not visible + not visible + no band visible blank

Second Row

Number: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Name: KB+ Ladder EcR pENT RXRLc Act2LacO 1 VP16 1 35s 1 35s 2 Act2LacO 2 Barnase 1 Barnase 2 CaMV Barstar Nost pENT 2 Barstar Nost pENT 1 KB+ Ladder
Expected Length(bp): 1008 500 630 1282 276 56 56 1282 ~250 ~250 515 1032 1032
Expected Length(bp): yes + yes + yes + yes, but with a second shorter band yes + yes + yes + yes, but with a second shorter band yes + yes + yes + yes + yes +


  • each successive image follows the gel running an additional 15 minutes after the initial 15 minute run

  • Summary: at least one copy of each part is good except for Act2LacO (which has the same old problem with the weird two bands) and LacIN-Nost-Act2LacO, which probably is a result of the fact that the LacO from which it was cloned has that weird band (and did at the time of cloning as well)

To Do

  • do a series of diagnostic digests on Act2LacO with different enzymes and also without any restriction enzymes
  • based on the results of that, prepare to re-ligate LacIN-Nost-Act2LacO (which I need as a whole part to do gibson on the whole fence)
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