IGEM:Cambridge/2008/Notebook/Voltage/2008/07/18

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Wet Work
There was no growth on the plates spread yesterday. There has been a few adjustments to the protocol.

Biobrick Extraction

Bigger, better, faster, stronger
We found we could not extract enough DNA from the registry, so we are upgrading the extraction to the "bigger, better, faster, stronger" method.

Warm 50μL of EB in Eppendorf tubes at 50°C and add 4 punched spots. Keep it warming at 50°C for 20mins and spin down for 3 minutes at 15,000 g. Warm agin for 10mins and spin down again for 3mins. Pipette off the liquid which should have the DNA in. We then confirmed with PCR.

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

And all 50μL were then run through the PCR reaction and 17μL of product run on an E-Gel with 3μL dye.

The results gave large amounts of DNA in each lane for all Biobricks except F. The original extracts of G,H,I,J,K and L 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

Prepare Agar plates : Add 200μL of Amp (100mg/mL) into 200mL of LA ( Final conc. of 100μg/mL ) and add 100μL of Amp (100mg/mL) into 200mL of LA ( Final conc. of 50μg/mL ) Plate neat samples of G,H,I,J,K,L onto both types of plate and incubate overnight.