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.

PCR tubes

The PCR tubes from the PCR were placed in a box labeled Split PCR DR + ZPS; 7/24/06 and placed in the 4 degree cyano-refrigerator.

Gels

• Two gels of the PCR products from a few hours ago were run at 22:30.
• The gels were 1% agarose.
  • There was a little bit of leakage of gel #2.
  • The agarose that leaked was cleaned up, along with the tray that contained the gel.
• 2um of 10x loading dye was added to 18um of PCR product and placed in the wells.
• 10um of ladder was added to both the first well seen and the last well.
• Gel 1 contains, in order: L, 1A, 2A, 3A, 1B, 2B, 3B, 1C, 2C, 3C, ZP(+), ZP(-), L.
• Gel 2 contains, in order: L, 4A, 5A, 6A, 4B, 5B, 6B, 4C, 5C, 6C, DR(+), DR(-), L.
  • Gel 2 was placed in one gel electrophoresis box which contained the message "buffer supplimented with 10mM MgCl₂", and was thereafter moved to a clean box with new TBE buffer.
    • Reasoning: The gel, when initially run, looked suspiciously like the time when the gel was overheated and crumbled on itself.
  • The loading dye used was BTB 50% glycerol 10x loading dye.
  • The gel was run for 55 minutes (the loading dye almost ran off)

Additional note

The door to the back room of 5088 was open; since the door says "Keep the door closed thank you," the door was indeed closed thank you.