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Troubleshooting from Monday

  • J37016 doesn't seem to have worked
    • One of the minipreps showed three bands, two corresponding to correct ligation, but one corresponding to an incorrect ligation.
    • We think that this is due to contamination (picking up 2 colonies from the plate)
    • The bacteria (liquid culture) were diluted out onto a plate and kept overnight
    • To do tomorrow: Pick 4 colonies to regrow and miniprep hopefully showing bands which we expect.
  • J37015RS - Dr. Jensen thinks that the ligation just didn't work, so we should attempt to redo the ligation tomorrow
    • Set up the digestion of the two parts 6B & RS->12D->24A
    • Run the gel overnight (keep to 5-10v)
  • 12D->2H - ligation hasn't worked for 4 times
    • Checked to see if there is a problem with the enzyme or maybe with the DNA itself.
    • Made 2 digests and control
      • 20 uL DNA digest with 1 uL enzyme PstI or SpeI
      • To be made up with water and yellow buffer
    • Gel run overnight with the reattempted ligation J37015RS
  • Dr. Jensen also would suggest that after every maxiprep, take over to Prof. Freemont's lab and obtain the concentration of DNA. For our amounts, the concentration of DNA should be around 100 ng/mL.
  • She also suggested that we sequence the following parts to ensure that we have done the ligations properly.
    • J37019 (9G->7A->3O) colony R (concentration 130 ng/uL)
    • RS+C0261+I13504 (RS->12D->24A) colony L (concentration 110 ng/uL)
  • To make up the sequencing solution
    • 1 ug DNA
    • 12.9 pM/uL primer
    • Make up with water to 12uL
  • More DNA ladder was supplied to us. Please note that this is a different DNA ladder than the lambda one we were using, so be sure to look at the lab wall with the fragment sizes to ensure you are reading it properly. Please only use 5 uL of the ladder at any one time for one lane in the gel.
  • More loading buffer was also obtained. Please only use 4 uL of loading buffer in any solution you want to get.
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