IGEM:Cambridge/2008/Protocols

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.

Primer Prep

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