IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-24

Morning meeting

• Western blots may prove very difficult; mass spectrometry is a possible alternative
• We'll retry sequencing of the first succesful genomic PCR product ("Lane 21"), since we were a bit sloppy with it last time
  • Make sure to send at least as much DNA as GeneWiz recommends (works out to 40-60 ng / µL for segments < 6kb)
  • Use our own M13 primers, not the universal ones that GeneWiz provides, since we're using a nonstandard Topo vector (Topo Blunt)
• We can slow down E. coli growth by using nutrient-diminished media, and thereby prevent KaiA/B/C from diluting as quickly

Game plan

• Inoculate more Hetmann Six from frozen stock, so we can miniprep it and send it off for sequencing
• Try a split PCR on the remaining Hetmann Six minprepped DNA (that is, PCR KaiA, KaiB, and KaiC separately, from the Topo + KaiABC plasmid)

Inoculations

• Grew each of HH 1-6 frozen stock in 8mL of LB + 3.2uL of Kan stock (50mg/mL, final concentration 20 µg / mL). Inoculated at 37C overnight.

Split PCR

• Did a PCR reaction to split KaiA, KaiB, and KaiC along with biobrick ends. Dave used template 1-3; Peng used template 4-6.

Using the Silver protocol:

 5 µL 10x ThermoPol buffer (Vent)
 1.0 µL 10 mM dNTPs 
 2.5 µL 20 µM forward primer 
 2.5 µL 20 µM reverse primer 
 1 µL plasmid DNA
 1 µL Vent DNA polymerase 
 37 uL dH20

PCR Time

#*95 °C for 2 min. (melt) 
#*95 °C for 0.5 min (melt) 
#*57 °C for 0.5 min. (anneal) 
#*74 °C for 2.0 min. (extension)
#* Go to step 2 (30x)
#*74 °C for 5 min. (final extension; modified from Silver protocol)
#* Keep at 4 °C forever

• Each person did 11 reactions; 3 for each of KaiA, KaiB, and KaiC, a negative, and a CAT gene positive. Peng's negative was with kaiB; Dave's with kaiA (no template added). Primers used can be found under primer design.
• Tubes are 0.2mL, colored in b/w with the number and color with the letter.

• Hetmann: It should be done around 6:40-7:00, assuming ramp time only takes 20-40 minutes. The machine is PCR6. 2 gels are siting there at 1%

PCR tubes

Some PCR tubes were left out on the counter, and there is some liquid inside. These tubes were moved to the 4 degree cyano-refrigerator.