IGEM:MIT/2006/Notebook/2006-10-31: Difference between revisions

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# Miniprepped vectors and digested to linearize.
# Miniprepped vectors and digested to linearize.
#Yeast cells didn't grow up overnight.
#Yeast cells didn't grow up overnight.
##This was unsuspected, as a parallel culture grown in the same media grew fine. I can only conclude that the slow growth was due to the face that I  
##This was unexpected, as a parallel culture grown overnight in the same media grew fine. I can only conclude that the slow growth was due to the fact that I innoculated from a freezer stock, which I have never done before and so don't know what to expect.
## I will transform the yeast tomorrow (Wed), and cut even more corners to get a culture ready for Friday.
==General to do list==
==General to do list==
*Streak and start overnight culture of pSB1AC3-J45250/pSB3K3-J45400 again.
*Streak and start overnight culture of pSB1AC3-J45250/pSB3K3-J45400 again.

Revision as of 15:52, 31 October 2006

Overnights

pSB1AC3-J45250/pSB3K3-J45400

pSB1AC3-J45250/pSB3K3-J45400 didn't grow.

pSB1AT3-J45700

Austin's pSB1AC3-J45700 didn't grow in Amp/Cm but did in Amp. Reshma's did (some).

pSB1AT3-J45400

  • Amp/Tet liquid culture didn't grow
  • did grow on amp plate
  • took colonies and innoculated into amp and amp/test media --Austin Che 11:29, 31 October 2006 (EST)

Austin's to do list

  • Miniprep cultures
    • Available cultures: 250, 320, 700
  • Digests:
    • ES: 250, 320, 700
    • XP: 180, 250, 400
    • EP plasmids: 2K4, 1AC3, 1AK3
  • Ligations:
    1. 1AC.320.180
    2. 2K|1AK.250.400
    3. 2K|1AK.700.250
    4. 2K|1AK.700.400
  • Transform into MachI
  • Pick J45180 colonies to grow o/n to glycerol
  • Send J45180 plate to registry

Reshma's assemblies

To make

  • pSB1AC3-J45320.J45180
  • pSB1AK3-J45250.J45400
  • pSB1AK3-J45250.J45700
  • pSB1AK3-J45700.J45400

Step 1

  • Cut J45250 with ES - started
  • Cut J45700 with ES - started
  • Cut J45700 with XP - started
  • Cut pSB1AK3-P1010 with EP - started
  • Cut pSB1AC3-P1010 with EP - started

Step 2

  • Purify ES cut J45250
  • Purify ES cut J45700
  • Purify XP cut J45700
  • Purify ES cut J45320
  • Purify XP cut J45180
  • Purify XP cut J45400
  • Phosphatase treat pSB1AK3-P1010
  • Phosphatase treat pSB1AC3-P1010

Step 3

  • Purify pSB1AK3-P1010
  • Purify pSB1AC3-P1010

Step 4

Ligations

Step 5

Transformations into TOP10 cells

Smell tests

  • Use 4.4μL stock IA-OH per 10mL culture
  • Use 40μL 0.5M SA per 10mL culture

These were started today

  • 100mL of each. In 500mL or 1L flask.

Dilutions

  • Used 1mL of pSB1AC3-J45200 (OD600nm=0.31) in each flask
  • Used 0.27mL of pSB1AT3-J45120 (OD600nm=1.11) in each flask
  • Used 0.27mL of pSB1AC3-J45250 (OD600nm=1.11) in each flask

Thus, each strain was diluted back to OD 0.003 in each flask.

Samples

  1. Coculture pSB1AC3-J45250 and pSB1AT3-J45120 without salicylic acid and isoamyl alcohol
  2. Coculture pSB1AC3-J45250 and pSB1AT3-J45120 with salicylic acid and isoamyl alcohol
  3. Coculture pSB1AC3-J45200 and pSB1AT3-J45120 without salicylic acid and isoamyl alcohol
  4. Coculture pSB1AC3-J45200 and pSB1AT3-J45120 with salicylic acid and isoamyl alcohol
  5. Culture pSB1AC3-J45250 without salicylic acid and isoamyl alcohol
  6. Culture pSB1AC3-J45250 with salicylic acid and isoamyl alcohol
  7. Culture pSB1AC3-J45200 without salicylic acid and isoamyl alcohol
  8. Culture pSB1AC3-J45200 with salicylic acid and isoamyl alcohol

These still need to be done

  1. Coculture pSB1AC3-J45250 and pSB1AT3-J45180 without salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AT3-J45180.
  2. Coculture pSB1AC3-J45250 and pSB1AT3-J45180 with salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AT3-J45180.
  3. Coculture pSB1AC3-J45250/pSB3K3-J45400 and pSB1AT3-J45700 without salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AC3-J45250/pSB3K3-J45400.
  4. Coculture pSB1AC3-J45250/pSB3K3-J45400 and pSB1AT3-J45700 with salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AC3-J45250/pSB3K3-J45400.
  5. Coculture pSB1AC3-J45200 and pSB1AT3-J45180 without salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AT3-J45180.
  6. Coculture pSB1AC3-J45200 and pSB1AT3-J45180 with salicylic acid and isoamyl alcohol
    • Couldn't start. No pSB1AT3-J45180.

Yeast Stuff

  1. Grew up overnights of ADH1promoter+BSGD in pRS306.
    1. Oddly enough, only 3 of my 9 overnights grew up. Those 3 had been innoculated with a ton of cells.
  2. Miniprepped vectors and digested to linearize.
  3. Yeast cells didn't grow up overnight.
    1. This was unexpected, as a parallel culture grown overnight in the same media grew fine. I can only conclude that the slow growth was due to the fact that I innoculated from a freezer stock, which I have never done before and so don't know what to expect.
    2. I will transform the yeast tomorrow (Wed), and cut even more corners to get a culture ready for Friday.

General to do list

  • Streak and start overnight culture of pSB1AC3-J45250/pSB3K3-J45400 again.
  • Make more LB media
  • Start overnight cultures of
    • pSB1AT3-J45120 (grows fast)
    • pSB1AT3-J45180 (do extra just in case)
    • pSB1AT3-J45250 (grows fast)
    • pSB1AT3-J45200 (do extra, grows slow)
    • pSB1AT3-J45700 (do extra, grows slow)
  • Autoclave some 250 mL flasks covered in foil

Done

pSB1AT3-J45400 submitted to Registry.

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