IGEM:Cambridge/2008/Notebook/Voltage/2008/07/18: Difference between revisions

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Each sample was then put through 34 cycles of PCR.
Each sample except the control was then put through 34 cycles of PCR.
The following were added to eppendorf tubes:
The following were added to eppendorf tubes:
5μL of DNA in EB buffer
5μL of DNA in EB buffer
2.5μL of each primer for Biobrick vectors
2.5μL of each primer for Biobrick vectors
25μL of Finnzymes mastermix
25μL of Finnzymes mastermix
15μL of sterile distilled water
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 the first.
The results gave large amounts of DNA in each lane for all Biobricks except the first.

Revision as of 11:28, 18 July 2008

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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

Class Registry Part Kit Location Tube Label
Promoter BBa_J23100 2008 1002-10E F
RBS BBa_B0030 2008 1002-5G G
Stop BBa_B1002 2008 1016-8A H
Stop BBa_B1006 2008 1016-8D I
Promoter BBa_J23113 2008 1002-12B J
Promoter BBa_J23114 2008 1002-12C K
Control pUC19 L


Warm 50μL of EB in Eppendorf tubes at 60°C for 30 minutes and add 4 punched spots. Spin down for 3 minutes at 15,000 g


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 the first. The samples G,H,I,J,K and L were then used for the following transformations.

Chilled 2μLmL 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.





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