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

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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.
    • In both gels, it seems that the initial well that was supposed to contain the ladder contains, instead, 10 um of dye
    • 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)
Split PCR to extract the KaiA, KaiB, and KaiC sequences independently from PCC7942, Gel 1

Click for legend
Split PCR to extract the KaiA, KaiB, and KaiC sequences independently from PCC7942, Gel 2

Click for legend
This is an image of a 1kb+ ladder, from the Kafatos webpage


Analysis:

  • Tough break, since all that is visible is a faint 5 kb band in three lanes (Gel1) and a under 100 bp segment, possibly primer dimers (since our smallest section of DNA, KaiB, is 309bp and the band is clearly not KaiB).
  • The gels were wrapped in seran wrap and placed in the 4 degree for further analysis tomorrow.

Additional notes

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.

Quote of the day: Reed: filter membrane choice important1

1. Freemantle, Michael, "From Barley To Beer in the Glass," Chemical & Engineering News, Volume 74, Number 46 pg. 36; ISSN 0009-2347

The Chinese food from lunch was left in the break room, and since I didn't have a good dinner, I decided to eat it all. Just joking, it looks pretty bad, and has been sitting out for 12 hours. Not sure what to do with it; I'll figure something out.