Team:Cambridge/Lab book/Week 11

Monday (03/09/12)

Tuesday (04/09/12)

Wednesday (05/09/12)

PCR products from Wednesday's PCR. Lanes 2 - 3: Fluoride riboswitch biobrick genomic DNA. Lanes 4 - 5: MGRS construct genomic DNA (+8). Lanes 6 - 7: MGRS construct genomic DNA (-8). Lane 8: MGRS biobrick genomic DNA (+8).

PCR products from Wednesday's PCR. Top: Lane 2: MGRS biobrick genomic DNA (+8). Lanes 3 - 4: MGRS construct backbone (expected size 9kbp) (+8). Lanes 5 - 6: MGRS construct backbone (expected size 9kbp) (-8). Lane 7: MGRS biobrick vector (+8). Lane 8: Positive control. Bottom: Lane 2: MGRS biobrick vector (+8). lane 3: Negative control.

PCR of biobrick fragments and MGRS construct fragments.

• Fluoride and magnesium riboswitch genomic DNA amplified with PCR, along with vector fragments for turning the MGRS into a biobrick and for producing the magnesium riboswitch construct.
• PCR settings: Annealing temperature - 58 °C, Elongation step - 60 seconds.
• Genomic fragments worked well, but longer backbone fragments mostly failed.
• Extractions of DNA from these gels failed almost completely. Will try purifying directly from PCR products next time - results may not produce such a high purity of the precise DNA we want, but the increase in yield should outweigh this problem.

Gibson assembly of -8 MGRS construct

• Fragments gel extracted earlier assembled with Gibson assembly.
• Fragments used:

• 9kbp fragment from tube 13 used as backbone.
• Genomic fragments from tubes 6 + 7.

Transformation of E.coli with Gibson products

• Gibson products from earlier today used to transform e.coli.
• Resultant transformants plated out on 100 μg/ml ampicillin plates.

Thursday (06/09/12)

Friday (07/09/12)

Saturday (08/09/12)

Gel for 9kb fragments of lux and flu construct:

File:Long frags PM 08.09.12.tif

Sunday (09/09/12)

Verification gel:

The results of our fluoride assay.