IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-10-22

Midiprep

3 midipreps: 011 (J04500 + KaiB), 010 + 012 (J04500 + KaiA + J04500 + KaiC), B00015 (terminator)

First two had succesful nanodrops (~60 and 90 ng/uL respectively), but B00015 did not turn out (flatline).

A fourth midiprep, of Geneart KaiB, is going extremely slowly. Right now it's on the first QC wash of step 11 in the Qiagen manual.

Digest

16 digests of 011\SP. Digesting for 3h at 45C in PCR-5 (imaging room). The digest should be finished at 7:20PM.

Reaction

• 7.6 uL DNA
• 1 uL 10x BSA
• 1 uL NEBuffer 2
• 0.2 uL SpeI
• 0.2 uL PstI

CIP'd vectors

Left set of 8 in freezer

Run on gel and gel extract

=( Bad results.... poststained, etc...

Gel results

"011" cut with /sp... or is it... it looks exactly like 022 cut with /sp..."

Diagnostic digest

50min digest @45. Lanes in the below gel

• 1: 1kb+
• 2: "11" undig
• 3: "11" /xp
• 4: "11" /sp jeff
• 5: "11" /sp
• 6: "22" undig
• 7: "22" /sp
• 8: "22" /xp
• 9: 1kb+

Note the quotes signify the old labeling. If you look carefully there is 500bp insertin lane 8, and 2 3000 bp fragments in lane 3; so the labeling was switched.

suspisions confirmed: the 2 samples were mislabeled =( Relabeled correctly"