Team:Cambridge/Lab book/Week 11

Monday (03/09/12)

Further Gibson diagnostics

No difference between PJs cells and my cells, or PJs master mix and mine, but he still obtains > 10x higher efficiencies.

Tonight, we both assembled with my positive control again, but this time used our plates and his plates to plate the cells, to check for any difference there.

Tuesday (04/09/12)

Further Gibson Diagnostics

Considered it might be the PCR machine. Theirs is a hot-block, ours is a hot-air/centrifuge - type. This allows them to transfer reactions from ice to 50 C rapidly by preheating the block, theoretically making them much better able to limit the activity of the T5 exonuclease (which should only chew away a couple of dozen bases from the 3', ideally).

Direct comparison of their PCR machine and ours for another assembly with our positive control.

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.

Further Gibson diagnostics

Direct comparison between PCR machines showed a >10x difference in efficiency. We have isolated most of our efficiency problems to our PCR machine.

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:

9 KB fragments