Jan 15, 2018
Colony PCR (DreamTaq) with Synechocystis sp. PCC 6803 and gel electrophoresis
- 1Institute for Synthetic Microbiology;
- 2Heinrich-Heine Universität Düsseldorf
- Axmann Lab
- CyanoWorld
Protocol Citation: Maximilian Dietsch, Miriam D 2018. Colony PCR (DreamTaq) with Synechocystis sp. PCC 6803 and gel electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.mk5c4y6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
I used this protocol in my bachelor thesis and it worked.
Created: January 13, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 9597
Keywords: Synechocystism agarosegel, colony pcr
Abstract
In this protocol, the extraction of template DNA out of Synechocystis sp. PCC 6803 samples, the PCR of the template and the application on a agarosegel are described. The protocol was handed over by Maximilian Dietsch MSc.
Safety warnings
Work under sterile conditions in the first step if you need your colonies for a further use.
Make sure to check your reagents and chemicals for safety warnings and correct waste disposal!
Before start
Culture your desired Synechocystis colonies for at least 7 days on BG11 agar plates.
You can use the recipe for standard BG-11 media, which includes the recipe for BG-11 media based agar plates, of Anna Behle MSc.
Always work under sterile conditions while inoculating your agar plates!
Inoculation
Inoculation
Inoculate 50µl sterile water in a tube with your desired colony.
It would be good if you use cell-material from a agar plate with an applied clone from an agar plate, so you have enough cell-material.
The inoculated water should have a fresh, light green colour.
Work under sterile conditions if you need your colonies on your agar plates for a further use.
50 µL nuclease-free water
Heating
Heating
Heat your samples up to 95 °C for 10 minutes.
Make sure that you don't burn yourself! The samples are hot!
95 °C
00:10:00
Centrifugation
Centrifugation
Centrifuge your samples at 13,000 rpm for 1 minute.
00:01:00
Pipette your samples
Pipette your samples
Pipette 1-2µl of the supernatant into a fresh PCR tube.
You can freeze the rest of your samples (including the pallet) at -20° C for a further use. Afterwards just thaw your sample by room temperature and centifuge it at 13,000 rpm for 1 min. Now you can pipette 1-2µl of the supernatant again for a later colony PCR.
Pipette your master mix
Pipette your master mix
Pipette your master mix. In this case, we use the Dream Taq DNA Polymerase Kit from Thermo Fisher Science.
Use the following contents for one sample:
10x DreamTaq buffer: 2µl
dNTP Mix: 0.4µl
Forward primer: 2µl
Reverse primer: 2µl
DreamTaq DNA Polymerase: 0.2µl
Water (nuclease-free): 12.4µl (for 1µl template) or 12.3µl (for 2µl template)
Total: 18-19µl
Pipette the Dream Taq DNA Polymerase in the end.
You can also use the Thermo Fisher DreamTaq Master Mix, which contains the buffer, dNTP Mix and the Dream Taq Polymerase. In this case, you just have to pipette The Master Mix,
the forward primer, the reverse primer. the template DNA and nuclease-free water. Use the related protocol from your DreamTaq PCR package leaflet.
Pipette your master mix to your sample
Pipette your master mix to your sample
Pipette 18-19µl of your master mix to your template DNA, which contains 1-2µl (from the previous steps).
Run your PCR
Run your PCR
Run a PCR with your samples.
Check the Tm of your primers to use the right annealing temperature. The DreamTaq Polymerase uses the annealing temperature which is 5 °C lower than your Tm.
Therefore I use a temperature that is 5 °C lower than my lowest primer Tm. For example: My lowest Tm is 63 °C. Therefore I use 58 °C as my annealing temperature.
You can also check your optimal annealing temperature with various mathematical equations or the Thermo Fisher Tm calculator.
https://www.thermofisher.com/de/de/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
In my case I use the following PCR protocol:
1. Initial denaturation: 98° C - 2 min.
2. Denaturation: 98° C - 30 sec.
3. Annealing: Tm - 5° C - 30 sec -> I use 58° C here.
4. Extension: 72° C - 1 min.
5. Final extension: 72° C - 10 min.
5. 4° C hold
Rerun the second to forth step for 35 times.
Note from Thermo Fisher:
The recommended extension step is 1 min. for PCR products up to 2kb. For longer products, the extension time should be prolonged by 1 min/kb.
01:12:00 PCR
Prepare your agarose gel
Prepare your agarose gel
Prepare an appropriate gel for gel electrophoresis.
I prepare an 1%, small agarose gel. Therefore put 0.5g of agarose in 50ml 1x TAE (TRIS-Acetat-EDTA) buffer.
Afterwards heat it up in a microwave with 360 watt until the agarose is completely dissolved and the solution is clear.
Cool it down at room temperature until it's hotter than lukewarm but still touchable and not burning your skin!
Shake it some times while it's cooling down and check the solution for lump formation. It should be clear!
Add your dye to your agarosegel. I use midoriGreen Advance with an 1:20,0000 dilution factor.
Therefore I pipette 2.5µl in my agarose gel solution.
Afterwards pour your prepared agarose gel in a gel carrier.
Don't forget to put your appropriate gel combs in your agarose gel before it hardens.
When your gel is hardened, take out your gel combs and put appropriate buffer in the gel chamber until the agarose gel is completely soaked.
Load your samples
Load your samples
Load your samples with an appropriate loading dye. I use the 6x loading dye of Thermo Fisher.
Therefore I pipette 4µl to each sample.
Load your gel
Load your gel
Pipette your samples carefully up and down to mix your samples with the loading dye.
Load your hardened agarosegel with your samples. Adjust the loading volume to the size of your gel comb.
Don't forget to use an appropriate ladder for your samples.
I use the Gene Ruler 1kb Plus DNA ladder (ready to use) to detect a fragment of a size of 708 bp.
Therefore I pipette 5µl of my ladder in one slot of the agarose gel.
Run your gel
Run your gel
Run your gel with your desired voltage. I use 120 V.
Adjust your running time to your fragment size.
I run my gel until it reaches 2/3 of the gel.
Check your results
Check your results
Check your results with your imaging system.
I use the ChemyDoc MP System of Bio-Rad to check my gel for my fragments. I use midoriGreen Advance to dye my fragments in the agarose gel.
Therefore I use the Nucleic Acid Gels -> GelGreen channel in my Image Lab 5.0 software.
Good luck!