Exeter/17 July 2013

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Revision as of 12:05, 19 July 2013

MiniPreps of yesterday's liquid cultures

The plasmids were extracted from our liquid cultures, giving us DNA from...

• CcaS, our green light sensor (BBa_K592001)
• CcaR, the intermediate protein which communicates between the green light sensor and the cI repressor system (BBa_K592002)
• New Part 2, a promoter and RBS added to FixJ, the intermediate protein which communicates between the blue light sensor and the yellow pigment (BBa_K608002 + BBa_K592005)
• Terminator, seven replicates (BBa_B0015)
• FixJ promoter, where FixJ binds to allow synthesis of yellow pigment (BBa_K592006)
• Magenta pigment (BBa_K592012)
• Yello pigment (BBa_K592010)
• Ribosome binding site (BBa_B0034)
• YF1, our blue light sensor (BBa_K592004)
• FixJ,the intermediate protein which communicates between the blue light sensor and the yellow pigment (BBa_K592005)

NanoDrop data

We found the concentration of DNA in each sample using a NanoDrop machine. Remember, we had seven replicates of our terminator (BBa_B0015)

- Terminator #1 - 3.5ng/ul

- Terminator #2 - 1.9ng/ul

- Terminator #3 - 30.5ng/ul

- Terminator #4 - 25.4ng/ul

- Terminator #5 - 2.4ng/ul

- Terminator #6 - 2.1ng/ul

- Terminator #7 - 15.0ng/ul

- CcaS - 94.0ng/ul

- RBS - 40.1ng/ul

- Magenta pigment - 45.5ng/ul

- Yellow pigment - 34.8ng/ul

- New Part 2 (promoter, RBS and FixJ) - 59.5ng/ul

- YF1 (blue light sensor) - 27.6ng/ul

- FixJ - 30.7ng/ul

- FixJ promoter - 20.1ng/ul

- CcaR - 4.9ng/ul

The data from the NanoDrop shows that most of our "important" parts have sufficient concentrations to go on to digestion, but CcaR has too low a concentration to be useful.

To increase the concentration of DNA per ul, the SureClean protocol was utilised. The concentration of CcaR went from 4.9ng/ul to 81.6ng/ul.

SureClean protocol

SureClean is a solution provided by bioline that allows column free nucleic acid purification. After a number of our digests we found the concentrations of resulting DNA to be unacceptably low when measured using the NanoDrop, fortunately SureClean provided us with a method of concentrating our samples.

Protocol:

1 - Add 6 ul of pink co-precipitate to your nucleic acid sample and mix well for 30s. For samples that are greater than 200ul the pink co-precipitate can be increased accordingly but it isn't ever necessary to use over 20ul.

2 - Add an equal volume of SureClean to your nucleic acid solution and mix well.

3 - Centrifuge on max speed (!4,000 x g) for 10 mins.

4 - Carefully remove supernatant by pipette.

5 - Add a volume of 70% ethanol that is equak to 2x the original sample volume and vortex for 10s.

6 - Repeat step 3.

7 - Remove supernatant and air dry to ensure all ethanol is removed.

8 - Resuspend pellet in the desired volume of TE, water or buffer.

9 - Use NanoDrop to measure concentration of resulting purified sample - hopefully it will have improved!

Second attempt at digestions

Our "Part A" BioBricks will be:

• CcaS
• Magenta pigment
• Yellow pigment
• New Part 2 (promoter, RBS and FixJ)
• YFI (blue light sensor)
• FixJ

Our "Part B" BioBricks will be:

• Terminator #3
• Terminator #4
• Terminator #5

Basically, we're adding terminators to our "important" BioBricks.

The digestion protocol from 11/7/13 was used, although a few changes were made. We are using FastDigest enzymes, which require less time in the thermocycler at 37_oC (generally 15 minutes maximum). Also, we will run the results of our digest on a gel to show that the enzymes (EcoRI, XbaI, etc) have cut in the right places. This denatures the enzymes, so we don't need the 80_oC stage in the thermocycler.

Running the results of our digestion on a gel