Day 1 Wet lab 22/06/15

Made LB plates

Made LB broth

TIPS autoclaved

Day 2 Wet lab 23/06/15

Set up Top10 overnight, VS45 overnight, US348 overnight

Meeting with advisors to discuss progress

Day 3 Wet lab 24/06/15

Filter sterilise the buffer

250ml no antibiotic broth, put top10 cells in

Incubate until optical density is 0.6

Miniprep PSB1C3 plasmid

Day 4 Wet lab 25/06/15

Transformation

Prepare SBC media

Digest of PSB1c3 from MS348

Day 5 Wet lab 26/06/15

Calculated competent cell efficiency

Agarose gel formation

Gel electrophoresis

Day 6 Wet lab 29/06/15

Transforming linear PSB1C3

Day 7 Wet lab 30/06/15

Miniprep of PSB1A3 plasmid

Gel electrophoresis of a large quantity of PSB1C3

Meeting with advisors to discuss progress

Day 8 Wet lab 01/07/15

Gel electrophoresis of the purified DNA extracted

Transformation of pSB1A3 circular plasmid to VS45 cells (competent)

Digest of all pSB1C3 plasmids

Day 9 Wet lab 02/07/15

Competent cells transformation efficiency

Mini-prep of 1MS340 to get pSB1C3 plasmid

Gel extraction of big digest

Quantification of the digest. Added sample buffer to each digest

Day 10 Wet lab 03/07/15

Transformation efficiency

Day 11 Wet lab 06/07/15

TOP10 cells containing pSB1A3 with limonene synthase were cultured overnight on AMP plates

Colonies of VS45 containing pSB1A3 were patched onto chloramphenicol plates

Overnight digest of miniprepped pSBIC3 using ECORI and PSTl

Day 12 Wet lab 07/07/15

Agarose gel of overnight digest - no bands were visible

Set up overnight cultures of pSBIC3 and pSBIA3 to be miniprepped the next day

Set up an overnight digest of pSBIC3

Day 13 Wet lab 08/07/15

Agarose gel of overnight digest - no bands were visible

Miniprep of pSBIA3 and pSBIC3

Overnight digest of pSBIA3 and pSBIC3

Day 14 Wet lab 09/07/15

Agarose gel of digested pSBIA3 and pSBIC3 - no bands were visible

Overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3 were set up, ready to be miniprepped

Day 15 Wet lab 10/07/15

Miniprep of plasmids pSBIA3 and pSBIC3

Day 16 Wet lab 13/07/15

Overnight cultures of MS349 containing pSBIA3 LIMS and MS340 containing pSBIC3 were set up, ready to be miniprepped

Overnight digest of miniprepped plasmids pSBIA3 and pSBIC3 using the enzymes ECORI and PSTI

Day 17 Wet lab 14/07/15

Agarose gel of digested plasmids pSBIA3 and pSBIC3 - it finally worked!!!

Miniprep of overnight cultures

Gel extraction of both plasmids

Analytical gel using Hyperladder I to quantify the plasmid

Overnight cultures of Top10 cells containing PVS72

Day 18 Wet lab 15/07/15

Miniprepped PVS72

Transformation of VS45 with PVS72

Made LB plates with Chloramphenicol and Ampicillin

Plated the transformed VS45 cells on the Chloramphenicol/Ampicillin plates

Agarose gel of leftover digested pSBIA3 and pSBIC3 from 14/07/15

Gel extraction of pSBIA3 and pSBIC3

Day 19 Wet lab 16/07/15

Counted the overnight colonies of VS45 with PVS72

Calculated the transformation efficiency