IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-30

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Contents

Ligation Protocol I used

Go to our Protocols section, extensive writeup.


Ligation #6 Plates

Thousands of colonies! Excuse the blurriness, used my cameraphone :(

Molar Ratio, 2uL ligation product
Molar Ratio, 2uL ligation product
Molar Ratio, 19uL ligation product
Molar Ratio, 19uL ligation product
Volume Ratio, 2uL ligation product
Volume Ratio, 2uL ligation product
Volume Ratio, 19uL ligation product
Volume Ratio, 19uL ligation product


Note that for 2uL we got a lot more colonies than for 19uL. Means that the ligation mixture is toxic to the cells; do not use 19uL anymore for chemically competent cells!

Finally

Did a colony PCR of 36 colonies + positive control, streaking each on part of a KAN plate. 8uL PCR supermix, 1uL VF2 (2nM), 1uL VR (2nM).


It looks like we've finally succeeded in ligating J04500\SP + KaiC\XP!

'Click here!

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Succesful ligations: V2 #4, V19 #1, #4, #5, #6, #7, M2 #7, M19 #2, #4, #8

Sequencing results for KaiB

... Don't make sense. Doing some AlignX work, shows that KaiB WITH biobricks prefix and suffix is in front of the J04500 promoter+RBS. Ideas?

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