Lab July 6 2009

Monday July 6, 2009

Goal today is to run columns and gels on two unknown cell cultures, one of which contains Puc18 and one of which contains Puc19

added 50 ug/ml RNAseA to the P1 buffer (supposed to be 100 ug/ml but Nano said that's about 5 time as much as is actually needed.)

Running the Qiagen Miniprep Protocol for plasmid preparation. Only significant difference is that we are keeping some of the supernatant after the N3 step for later analysis if our gel fails for some reason.)

Purchased a bag of 1.5 ml epi's on Fran's account (must be returned when we have money!)

spinning at 8,000 rpm for 10 minutes to pellet cells from culture media, doing 4x 1.5 ml, 2 of each culture, b and c

Using 1.5ml epi tubes for the initial steps of the column does not work. There isn't enough room to clear the buffer through the column. We have gone to using the original collection tubes and using a sterile 1.5ml epi for the final collection step.

Ran a gel: Agarose Gel Electrophoresis Protocol

lanes 2, 3 - Sample B

lanes 6, 7 - sample C

stained gel in gelred, then imaged (shortened stain period to 10 minutes. Getting towards the end of the day. This, plus a perhaps overly thick gel, reduced the strength of the bands significantly but they were still visible. We are to always run a ladder with our gels. Nano will get from Allison tomorrow and nanodrop then sequence them.)

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