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Exeter/17 July 2013
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The plasmids were extracted from our liquid cultures, giving us DNA from... | 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) | ||
| - | + | We found the concentration of DNA in each sample using a NanoDrop machine. Remember, we had seven replicates of our terminator (BBa_B0015) | |
| - | - New Part 2 | + | - 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 | ||
==SureClean protocol== | ==SureClean protocol== | ||
Revision as of 10:50, 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)
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
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!
