IGEM:Cambridge/2008/Protocols
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Biobrick Extraction
We found we could not extract enough DNA from the registry, so we are upgrading the extraction to the "bigger, better, faster, stronger" method.
Spot Removal
- Warm 50μL of EB in Eppendorf tubes at 50°C and add 4 punched spots.
- Keep it at 50°C for 20mins
- Spin down for 3 minutes at 13,000 g.
- Warm for 10mins
- Spin down again for 3mins.
- Pipette off the supernatant which should contain DNA.
We then confirmed with PCR.
PCR Mix
Each sample except the control was then put through 34 cycles of PCR. The following were added to Eppendorf tubes:
- 5μL of DNA in EB buffer
- 2.5μL of each primer for Biobrick vectors
- 25μL of Finnzymes mastermix
- 15μL of sterile distilled water
- 50μL were then run through the PCR reaction
- 17μL of PCR product run on an E-Gel (0.8%) with 3μL dye.
The results gave large amounts of DNA in each lane for all Biobricks except F.
Transformation
The original extracts were then used for the following transformations.
- To chilled tubes add 5μL of DNA + EB solution to 5μL of Cells ( Chemically competent Top 10 )
- Ice for 30 minutes
- Heat shock at 42°C for 60 seconds
- Ice for 2 minutes
- Add 500μL of SOC
- Incubate at 37°C overnight
Plate Out
Prepare Agar plates :
- Add 200μL of Amp (100mg/mL) into 200mL of LA ( Final conc. of 100μg/mL )
- Add 100μL of Amp (100mg/mL) into 200mL of LA ( Final conc. of 50μg/mL )
- Plate neat samples of onto both types of plate and incubate overnight.
Flame Photometry
Set Up
- Make sure discharge pipe (on righthand side) is placed in something to collect discharge, and valve is not completely shut.
- Check clear tube is connected to compressor OUTLET, and securely attached.
- Turn on compressor.
- Gauge on back of photometer should read 12psi.
- Check gas pipe is attached at tap.
- Turn on gas.
- Wait 1 minute.
- Turn on photometer. It should click, and orange "flame on" light should come on.
- Place beaker of distilled water in the photometer tray, and make sure thin tube reaches water.
- Wait 15 minutes before taking any readings.
- Use "blank" dial to adjust reading to 0 for distilled water.
Taking readings
- Place thin tube into sample, far in, as the liquid will be taken up quite rapidly.
- Wait for reading to stabilise (reading may continue to fluctuate at high values) and take reading.
- Place tube in distilled water beaker between each sample, waiting until reading returns to 0.
In case of compressor blow-out
- Turn off gas at tap.
- Turn off photometer.
- Turn off compressor.
- Redo set up steps, with 15 minute waiting time reduced to 5, as the equipment will already be almost up to temperature.
Agarose Gel
Used for Electrophoresis when extracting any product afterwards.
Making the Gel
- Agarose Gel is made by adding the appropriate mass of Ultrapure Agarose (usually 0.8g) to a duran bottle, and then adding 100ml of TAE buffer to the bottle. 100μL of SyBr Green (10000x) can be added if necessary.
- The whole is then microwaved at high for 1 minute, stood for 3 minutes and again heated for 1 minute. Boiling should be prevented and the powder should fully dissolve, and then be left to cool.
- The ends of the gel tray must be taped with autoclave tape and firmly sealed.
- When the solution is hand hot the gel can be poured. Pipette around 1ml of the solution up each of the taped ends of the gel tray to prevent leakage. Insert the comb and pour the gel to around 6ml.
- Leave to set and remove the comb and tape.
Loading the Gel
- Mix around 17μL of DNA sample with 3μL of loading dye. The exact amount of DNA varies according to solution strength, but should be around 80ng
- Place the gel tray with gel into the running bay (wells at black end) and add enough TAE buffer to just cover the gel.
- Carefully load around 20μL of sample into wells, including some ladder as reference. Normal Agarose gels do not need to have every well filled - there is TAE in them already.
Runnig the Gel
- Connect up the red lead to the red terminals and the black lead to the black terminals of the power supply and the running bay.
- Make sure the wells are at the black end.
- Set the power supply to 80 volts and switch it on.
- Check the gel every 20 mins or so to make sure that the gel is running the right way and that the yellow dye (equivalent to 60bp) has not run to the end of the gel.
- When the front dye has reached about 3/4 of the way to the end of the gel turn the power supply off and disconnect the leads.
Visualising the Gel
- If no SyBr green has been added the gel needs to be placed in the EtBr solution for 20 mins.
- Drain the gel and place in the UV visualiser.
- If extracting make sure the prep UV button is pressed, and take a picture of the gel.
- To extract the gel slide the light tray out, ensure shielding for all who are involved, and carefully cut out the gel fragment, usind prep UV.
Gel DNA Recovery (Zymoclean)
- Add 3 parts ADB Buffer to 1 part Gel volume
- Incubate 55°C for 5-10 minutes, until gel melts
- Place solution into Zymo-Spin column and then into a collection tube
- Spin for 30 seconds. Empty collection tube when necessary
- Add 200μL Wash Buffer. Spin for 30 seconds. Repeat wash step.
- Place column into Eppendorf. Add 6-10μL water to elute DNA.
Plasmid Miniprep (Zyppy)
- Add 100μL 7x Lysis Buffer to 600μL Cell Culture. Invert 4 to 6 times
- Add 350μL cold Neutralization Buffer. Invert 4 to 6 times
- Centrifuge 13,000g for 2 minutes
- Transfer supernatant to Zymo-Spin column
- Place column in a collection tube and spin for 15 seconds. Discard flow through
- Add 200μL Endo-Wash Buffer. Spin for 15 seconds
- Add 400μL Zyppy Wash. Spin for 30 seconds
- Transfer column to clean Eppendorf. Add 30 to 100μL to the column and spin for 15 seconds to elute DNA
Primer Preparation and Storage
Sigma Primers arrived as tubes of dried oligos
- Suspend in sterile distilled water (SDW) to form a 100μM stock solution - stored at -20°C
- 10μM aliquots taken using 10μL stock and 90μL SDW - stored at 4°C
