Team:UCLA/Notebook/Protein Cages/24 July 2015
Intro: Second round of PCR to add on iGEM suffix. Ran on agarose gel to confirm. Didn't work (300 bp bands). Sad day :(
PCR: Gradient PCR at temps of 58, 62, 66, and 70 C using Q5 polymerase. Followed NEB Q5 protocol. 2.5 uL of 2 mM dNTP's. 0.25 uL of 1 ng/uL cage template. 14.5 uL water per reaction.
Agarose Gel: Used gel Phil made that was stored in 7731 fridge. Leftmost lane (#1) generuler 1 kb ladder. Lane 2 empty. Lanes 3-6 58, 62, 66, 70 C from left to right. Media:IMG_2602.JPG
Conclusion: Need to figure out improper annealing problem.
Ordered a second prefix primer to anneal to the handle, Eco site, and part of the Not site on the prefix. Sequence: ACTGACgaattcgcgg
Tyler Lee --Wtleeiv 18:19, 24 July 2015 (CDT)