Week 6

08/09/15

• Miniprepped the liquid cultures from lldd_ IDT, 4 tubes.
• Resuspended RFP in PCSB1C3 backbone (shipping plasmid) from distribution kit in 10uL nuclease-free water ( Plate 4, well 4B)
• Transformed 2 tubes with RFP backbones to amplify, 4uL plasmid per tube.
• Plated on 2 plates, 100uL mixture per plate. The plates were plates in the incubator
• Also performed the digestion and ligation : Promoter + LLD on AmpR.
• 4 separate tubes of ligation product, labelled P+LLD ligate 1-4 is in the freezer box
• tubes 1 and 2 were ligated to Amna’s AmpRbackbone
• tubes 3 and 4 were ligated to Amna’s AmpRbackone 2

09/09/2015

• Transformed Pro +lldd contruct ( AmpR). The 4 plates are in the incubator
• Miniprepped the Pro+ TnaA contruct ( KanR). The 6 tubes are in the freezer box, the average concentration is 20ng/uL

Digestion And Ligation

• We followed the standard digestion and ligation protocol
• Join Pr +TnaA to TnaB + Ter and put on both pSBC13 and AmpR backbone

PHOTO

• Used tubes 4,5,6, from Pro+TnaA miniprep from 9/09
• We halved the volumes of digestion procedure to give total digest volume of 25uL.

10/09/2015

• Picked the colonies of the tnaA and lldd and inoculated the final construct on AmpR backbone and shipping plasmid

11/09/2015

• Miniprepped the final construct on AmpR backbone and shipping plasmid

12/09/2015

• Switched the final construct of tnaA and lldd onto shipping plasmid

Week 7

13/09/2015

• Transformation of shipping constructs lldd and tnaA onto shipping plasmid using previous standard protocol
• Created 4 replicates for each

14/09/2015

• picked the white colonies and inoculated the liquid cultures following standard protocol

15/09/2015

• miniprepped the final construct for shipping