Team:Hawaii/Meeting/2008-07- 3

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Contents

Agenda

  1. Question to all: publicity photos with Vice Chancellor and Graduate Dean?
  2. Grace/Krystle: Timeline for part B (secretion)
  3. Margaret: Methods for part A (plasmid)

Minutes

Present: Krystle, Grace

  1. Plan B Timeline:
  2. Professor advice:
    • do step 7 with super competent cells, sequence it
    • are BamHI and HindIII compatible with buffers? May need to look at this step closely

Advice for Group:

  1. Run the iGEM proposals by the professors to see if it is really polished.
  2. We need to order some more competent cells, get a bigger batch, want to do it next week
  3. Our death resistant cells are coming, along with the lux
  4. Transformation advice:
    • Don't leave cells more than 5-10 minutes on ice
    • Getting only a few transformants is odd, should get lots or none
    • Try doing shaking step in 13mL vial so aeration occurs
  5. Test out ligation and see if we get transformants. May need to do an efficiency test again (compare super competent cells vs. home-made)
  6. Plasmid Prep advice:
    • use the cheese cloth, don’t bother to autoclave
    • get a DNA prep that is totally free of RNA, linearize, make sure you can get clean gels so you don’t lose small stuff
    • phenol/chloroform really gets rid of protein, we probably have lots (protein will affect the restriction digest) shake for 3-4 minutes, put on polypropylene rack, cover and shake it
    • fuzzy bands may mean salt contamination
    1. buffer too old? Will be apparent on marker and sample
      • salt in gel?
      • TE should not be a problem
    • the reason for the 70% ethanol wash is to get rid of salts (this is the problem)
    • stuff at top of wells means chromosomal DNA: too rough with cells when they are broken open: invert only! otherwise you will sheep DNA, plasmid stays in solution based on size and not associated with
  7. Gel:
    • For 9kb plasmid, use 0.8% gel
    • always rinse the comb
    • when re-using gel, make sure you are not losing water weight each time; may need to add more

if gel isn’t stained, you can restain it. over night in the refrigerator with Ethidium bromide, so bands won’t diffuse. can’t sequence until you get clean DNA Important to do for this week:

  1. Get the last two biobricks we need.
  2. Get a plasmid prep that is clean
    • free of RNA: add at a later step and incubate 1.5 hours
    • free of protein: use the phenol/chloroform
  3. Use the plasmid we have, run on a gel, so won’t need to clean it up

cut plasmid separately with the re enzymes, make sure it is inefficient: Lane 1: uncut lane Lane 2: with one enzyme Lane 3: with the other Lane 4: empty Lane 5: empty Lane 6: cut with both

  1. Overlapping oligonucleotides experiment: run products on a 4% gel to monitor progress.

Advice for Margaret:

  1. Re-do the PCR from 6/30.
  2. Sequence rep band from PCR7/2 (see Follow-up Experiments Section).
    • To do this, I have to re-do the PCR, clean-up, then send for sequencing.
  3. See if I am amplifying something else.
  4. Some stuff does not amplify with accusure. Try the rep region with another type of taq.
    • For this reaction, try several different dilutions of the template
  5. check:did they sequence pRL1383a?
    • priming sites may not even be there
    • design primers so that we can verify the existence of my priming sites.

Action Items

  • <Person A>: Task

Coming Up


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