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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, 19uL ligation product
Volume Ratio, 2uL 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!


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!

click for caption
click for caption

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?