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From 2013.igem.org

Morning

Today we will be trying to digest and ligate some of our BioBricks for the first time!

MiniPrep

We extracted the plasmids for...

- BBa_K608002, a promoter and RBS - BBa_K592005, our FixJ protein intermediate - BBa_K592004, our blue light sensor - BBa_B0015, a terminator

The normal MiniPrep protocol was followed, however the BBa_K608002 and BBa_B0015 Bricks only had 2/3rds as much genetic material collected as the other Bricks. (Plasmids collected from 2 Eppendorfs, as opposed to the usual 3)

Digestions

We are combining...

- a promoter and RBS to the blue light sensor (BBa_K608002 + BBa_K592004)

- a promoter and RBS to the FixJ protein coding region (BBa_K608002 + BBa_K592004)

- the magenta pigment to a terminator (BBa_K592012 + BBa_B0015)

- the yellow pigment to a terminator (BBa_K592010 + BBa_B0015)

First we have to work out the concentrations of each plasmid using a NanoDrop machine.

NanoDrop results

For our promoter and RBS Brick (BBa_K608002) and terminator Brick (BBa_B0015) we have two replicates.

- Promoter and RBS #1 - 29.1ng/ul

- Promoter and RBS #2 - 25.2ng/ul

- Terminator #1 - 27.6ng/ul

- Terminator #2 - 19.2ng/ul

- Blue light sensor - 38.3ng/ul

- FixJ intermediate - 58.1ng/ul

- Yellow pigment - 45.7ng/ul

- Magenta pigment - 72.0ng/ul

- RFP control - 43.4ng/ul

Ligate

• K592010 and B0015
• K592012 and B0015
• K592003 and Q04510
• K592005 and B0015
• K592002 and B0015
• K592001 and B0015
• K592004 and B0015