IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-31

Monday meeting

We've decided to refocus our efforts on preparing for the E. coli experiments. Our synthesized constructs will arrive on August 8 and we want to hit the ground running.

Split-extraction PCR #1

This PCR will extract KaiA, KaiB, and KaiC individually from the cyanobacteria genome. This should save us a step compared to extracting KaiABC as one segment and splitting them later. Also, if this PCR succeeds and produces 850bp, 300bp, and 1550bp bands, we'll have very high confidence that we've actually obtained template.

Samples

We'll use 2 samples, prepared as follows:

LC3A

• 1 mL from PCC 7942 flask (2006-7-16, reinoculated from liquid culture of 2006-7-7)
• Spun for 1 minute at 14k RPM
• Discard supernatant, resuspend pellet in 150 µL H2O.
• Lyse for 5 minutes at 95C.

LC3B

• same as LC3A, with one more centrifuge + resuspension cycle.

Reactions

All reactions will be 100 µL total volume. The mixture is as follows:

• 1 µL template
• 10 µL buffer
• 2 µL 10 mM dNTP
• 2 µL primer1
• 2 µL primer2
• 1 µL Vent polymerase
• 82 µL H2O

Reaction #	Sample	Primers
1	LC3A	exAF, exAR
2	LC3A	exBCF, exBOR
3	LC3A	exCOF, exBCR
4	LC3B	exAF, exAR
5	LC3B	exBCF, exBOR
6	LC3B	exCOF, exBCR
7	LC3A	none
8	LC3B	none