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

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Contents

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 1Click for legend
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 2Click 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
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.
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