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iGEM Bioware 

iGEM Bioware . 7.12-16.2010. Arsenic  . PCR - 62% Successful! We're still having trouble with LamB and ArsB. Ligations and Transformations have been unsuccessful.  Anything we can change to make these procedures more     effective?. Cloning parts from E. Coli Genome. pArsR F2R1. Neg.

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iGEM Bioware 

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  1. iGEM Bioware  7.12-16.2010

  2. Arsenic   PCR - 62% Successful! We're still having trouble with LamB and ArsB. Ligations and Transformations have been unsuccessful.  Anything we can change to make these procedures more     effective?

  3. Cloning parts from E. Coli Genome pArsR F2R1 Neg. Cont. pArsR F1R1 1kb  ladder LamB F1R lamB F2R  ArsR Fusion ArsR pArsR F1R2 pArsR F2R2

  4. Cloning parts from E. coli genome Digestion of Plasmid Backbones    1kb   Tet (X,S)   Tet (E,S)   Cap(X,S)  Cap(E,S)  1kb

  5. Gold This week involved a lot of PCR troubleshooting - Tried different amounts of template: 0.5, 1, 2, 5, 10 ul (10 - 200 ng) and less primer (2 uM) - Tried different annealing temperatures with a PCR gradient - Need to try everything with fresh template from genomic prep and with a positive salmonella PCR control We also have attempted digestion/ligation/transformation a few times with no results (lost too much DNA during PCR purification and gel extraction) - However, possibility that golS was successfully ligated into backbone and transformed into E.coli     - PCR for confirmation this morning

  6. Decoder -mFOLD -sRNA     -MicA     -MicF     -GadY -Hfq -Coliroid

  7. mFOLD (MicA) Energy Diagram

  8. mFOLD (MicA)

  9. mFOLD (MicA Biobrick)

  10. Composite Parts (colony PCR)

  11. Issues -Still working on SDM (Hfq) -TetR is still unsuccessful

  12. Questions Is there any way we can increase yield on PCR clean ups? or gel extractions? What is the band we're seeing in the lanes for lamB? How do the 260/280 and 260/230 ratios affect our sample?  Are there any remediatory procedures to correct bad ratios? When are we meeting for GAMES camp?

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