Exeter/17 July 2013

From 2013.igem.org

(Difference between revisions)
(SureClean protocol)
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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...
-
- BBa_K592001, our green light sensor (CcaS)
+
*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)
-
- BBa_K592002, which codes for the protein (CcaR) which communicates between our green light sensor and inverter gene which eventually regulates transcription of the magenta pigment gene
+
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, which has a promoter and RBS added to the FixJ coding region (BBa_K608002)
+
- 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!