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Exeter/17 July 2013
From 2013.igem.org
Fentwistle (Talk | contribs) (→Running the results of our digestion on a gel) |
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*Terminator #5 | *Terminator #5 | ||
| - | Basically, we're adding terminators to our "important" BioBricks. | + | Basically, we're adding terminators to our "important" BioBricks. We have also decided to digest the FixJ promoter and RBS to confirm that the digest protocol is working. |
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<sub>o</sub>C (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<sub>o</sub>C stage in the thermocycler. | 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<sub>o</sub>C (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<sub>o</sub>C stage in the thermocycler. | ||
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==Purifying the DNA from our gel== | ==Purifying the DNA from our gel== | ||
| - | We | + | We expected to see a line of bands running across the gel showing the various plasmids our Parts come in. Each lane would also have a second band representing the Part which had been cut out of the plasmid. The terminators, FixJ promoter and RBS are all very small lengths, so could potentially run off the gel (which appears to have happened in the gel image above). |
| + | |||
| + | You can see from the image of the gel that our digestion appears to have worked. We have two bands per lane for CcaS, "New Part 2", FixJ, YF1, magenta, yellow and our RFP control. | ||
| + | |||
| + | We cut the bands which don't represent a plasmid out of the gel using a scalpel, then extracted the DNA from the gel slices using a gel purification kit. | ||
Revision as of 15:37, 19 July 2013
Contents |
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)
- Yellow 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. We have also decided to digest the FixJ promoter and RBS to confirm that the digest protocol is working.
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 37oC (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 80oC stage in the thermocycler.
Running the results of our digestion on a gel
We also tried a different way of running the DNA on the gels. The actual gel itself is the same, and we are still running 10ul of ladder (HyperLadder 1), but for the actual DNA, we have 18ul of each Part A/B (listed above) with 3ul loading dye (6X DNA Loading Dye).
We expected to see a line across the gel of the various plasmids the Parts come in, and then a second line for each Part. The terminators, FixJ promoter and RBS
INSERT IMAGE OF GEL HERE (no pressure, Beth!)
Purifying the DNA from our gel
We expected to see a line of bands running across the gel showing the various plasmids our Parts come in. Each lane would also have a second band representing the Part which had been cut out of the plasmid. The terminators, FixJ promoter and RBS are all very small lengths, so could potentially run off the gel (which appears to have happened in the gel image above).
You can see from the image of the gel that our digestion appears to have worked. We have two bands per lane for CcaS, "New Part 2", FixJ, YF1, magenta, yellow and our RFP control.
We cut the bands which don't represent a plasmid out of the gel using a scalpel, then extracted the DNA from the gel slices using a gel purification kit.
