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.

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

1kb ladder

LamBF1R

lamBF2R 

ArsRFusion

ArsR pArsRF1R1 pArsR

F1R2

pArsRF2R1 pArsR

F2R2

Neg.Cont.

   1kb   Tet (X,S)   Tet (E,S)   Cap(X,S)  Cap(E,S)  1kb

Digestion of Plasmid Backbones

Cloning parts from E. coli genome

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

Decoder

-mFOLD-sRNA    -MicA    -MicF    -GadY-Hfq-Coliroid

mFOLD (MicA)Energy Diagram

mFOLD (MicA)

mFOLD (MicA Biobrick)

Composite Parts (colony PCR)

Issues

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

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?