Thursday, January 29, 2015

bio125, Thu, 20150129, mini prep, plasmid map, ApE

Section 1

8-8:20am. review mutation assignment
DNA mutation assignment, Wikipedia is wrong.

review mini prep protocol ApE

absorption spectrum of DNA and protein

Endo wash is to improve transformation efficienty (remove endotoxin, endonuclease. Though this is probably not a problem for yeas, Kioko found it improve 260/280 ratio).

Student asked why the elution buffer is used as blank control to measure DNA concentration.

9am. mini prep lab started.
by 10am. Students finished first round of centrifuge (binding DNA to zappy columns)

11am. Some students came back from nano drop measurement. DNA yields and 260/270 ratio were good.

Students not sure whether they should change new tips every time!
One student hold micropitte with tip upside down
Most students do not know to how resuspend properly. In large falcon tubes, Kioko prefer to use pipette up-and-down.
Many students are clear when lysis will be done.
Many students did not racks when they pick tubes from centrifuge.
Students did not realize there were two micro-centrifuge in room 351.

Tips: Kioko gave TE, lysis, neutralization buffer, wash buffer, elution buffer separately but in order in order to minimize mixups of tube. (This seems to slowed things down, but avoided chaos)
Kioko asked students to one additional spin to dry the columns. 

Section 2:
15 min review preclass lab assignment
30min, student went through protocol
10 minutes, show video from morning section

by 1:55 pm, miniprep started.

by 3:09 pm, most groups finished eluting plasmid DNA. Students went to 2nd floor core facility for nanodrop DNA concentration measurement.

by 4pm. All groups finished nanodrop measurement. So, nanodrop took 1 hour.

Similar problems with section 1.
A group arranged 15ml tubes in unbalanced positions.

Kioko thinks elution buffer should be increased to 50 ul, given that we used 5ml Ecoli.

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