IGEM Bioware 7.12-16.2010. Arsenic PCR - 62% Successful! We're still having trouble with LamB and...
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Transcript of IGEM Bioware 7.12-16.2010. Arsenic PCR - 62% Successful! We're still having trouble with LamB and...
![Page 1: 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.](https://reader035.fdocuments.us/reader035/viewer/2022062422/56649efa5503460f94c0c419/html5/thumbnails/1.jpg)
iGEM Bioware
7.12-16.2010
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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?
![Page 3: 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.](https://reader035.fdocuments.us/reader035/viewer/2022062422/56649efa5503460f94c0c419/html5/thumbnails/3.jpg)
Cloning parts from E. Coli Genome
1kb ladder
LamBF1R
lamBF2R
ArsRFusion
ArsR pArsRF1R1 pArsR
F1R2
pArsRF2R1 pArsR
F2R2
Neg.Cont.
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1kb Tet (X,S) Tet (E,S) Cap(X,S) Cap(E,S) 1kb
Digestion of Plasmid Backbones
Cloning parts from E. coli genome
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GoldThis 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
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Decoder
-mFOLD-sRNA -MicA -MicF -GadY-Hfq-Coliroid
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mFOLD (MicA)Energy Diagram
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mFOLD (MicA)
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mFOLD (MicA Biobrick)
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Composite Parts (colony PCR)
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Issues
-Still working on SDM (Hfq)-TetR is still unsuccessful
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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?