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Ligation #6 for Peng

This time, using marked 8.29.06 J04500 /sp in a pink tube and 8.29.06 KaiC /xp in a green tube. These are both pretty concentrated, and have the advantage of not having EtBr ever or UV exposure, as well as an additional PCR wash step. Both are eluted in EB.

Using the whole ligation mixture is still questionable to me, especially now as we have concentrated DNA. Also although Nick and Perry have been using the 1:3 volume ratio (and have had limited success), that seems like it's not a very good idea to me. With good nanodrop values I will continue using a 1:3 molar ratio, and do a sample using the 1:3 volume.

Regarding the control: I know ideally this should have a control to see if EtBr does affect ligation efficiency, but as I see it as we don't have any colonies from our previous experiments using equally dense DNA (although the #5 attempt may have had a bloated J04500 value from the gel ran yesterday), it'd be very unlikely that a control would produce colonies while avoiding EtBr would cause none to appear. Knowing that we haven't been able to get any colonies using EtBr, I think if no EtBr gave us colonies that would be signifigant enough.

Regarding Roche: Called, they said that the DNA dilution buffer was proprietary but one should use 2ul of the 5X stock + 8uL DNA to add to 10uL. Checked the pH of the Dilution Buffer, and it either is set around 7 or actually is not a pH buffer at all.

Experimental Setup:

  • 1:3 molar
    • 82ng J04500/sp @ 82ng/uL = 1 uL
    • BB insert: 3*(~2100/3400)*100 = 151.94 ng @ (60 ng/uL) = 3.37 uL
    • 5x DNA Dilution Buffer = 2uL
    • dH20 = 3.63 uL
      • Pipet 2uL into 37.5uL colony
      • Pipet 19uL into 37.5uL colony
  • 1:3 volume
    • 2uL vector
    • 6uL insert
    • 5x DNA Dilution Buffer = 2uL
      • Pipet 2uL into 37.5uL colony
      • Pipet 19uL into 37.5uL colony
  • 15uL Neg
  • 15uL positive (2uL puc19)

(3 vials of Top10 needed)

Let ligation sit for 1 hour, add DNA to cells, sit on ice for 30min, Heat shock 42C@30s, ice recovery for 2min, SOC for 1h.

Plated on KAN, except for the positive control which was on carb.

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