Glasgow/Wetlab/Week9

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!Fragments If 5'-3'
!Fragments If 5'-3'
!Fragments If 3'-5'
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!Result
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|(*a→d*)
|(*a→d*)
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|2025bp, 4803bp
|2025bp, 4803bp
|2595bp, 4232bp
|2595bp, 4232bp
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|-
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|(*d→g*)
|(*d→g*)
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|2843bp, 249bp, 4256bp
|2843bp, 249bp, 4256bp
|3686bp, 3413bp, 249bp
|3686bp, 3413bp, 249bp
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|}
|}
</ol>
</ol>

Revision as of 18:51, 30 August 2007

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Contents

Week 9

Monday 27th August 2007

  1. Maija performed a PvuI digest for pDntA+3/20G and pDntA+4/6B constructs with Roche's PvuI enzyme. The bands were weird sizes, see the gel here.
    Insert Vector Enzyme Expected size Result
    pDntA 3/20G PvuI 1450bp + 749bp ~2500bp
    pDntA 4/6B PvuI 2375bp + 681bp ~3800bp + ~1250bp
  2. Christine digested minipreps of interest B, C and D suspected to contain (*d→g*) from 24/08/07 with PstI. Miniprep of colony D shows bands of expeceted sizes 1155bp, 549bp, ~1962bp, and 3682bp.
  3. Overnights were also set up of colonies K, L, M, N, Q, S and T, each suspected of containing (*a→d*), from plate 2.

Tuesday 28 August 2007

  1. Lynsey, Maia and Christine did minipreps of overnights of colonies H, I, J, K, L, M, N, O, P, Q, R, S, T, U and V from Plate 2. Christine then ran a gel of the minipreps and K, L, M, N, Q, S and T showed bands interest of ~6.7kb.
  2. Christine digested all minipreps of interest for (*a→d*) and miniprep D of (*d→g*) with EcoRI. However, none showed bands of interest - possibly a fault of the enzyme, new EcoRI ordered.
    Insert (*a→d*) (*d→g*)
    Enzyme EcoRI EcoRI
    Fragments If 5'-3' 928bp, 2227bp, 18bp, 3654bp 2997bp, 18bp, 3654bp, 679bp
    Fragments If 3'-5' 928bp, 5597bp, 18bp, 284bp 679bp, 284bp, 18bp, 7046bp

Wednesday 29th August 2007

  1. Scott did colony PCR (see protocol 9) on the assembly transformations carried out yesterday using primers VF2 and VR. Unfortunately it looks as though the combined product is larger than the sum of its parts...
  2. Scott also did the following restriction digests (see Protocol 7):
    Insert Digest 1 (EcoRI/PvuI) Insert Digest 2 (EcoRI/SpeI)
    (Done overnight at 16°C)
    J23119 (tube 1) J61101
    J23119 (tube 2) "


    Destination (EcoRI/XbaI)
    (Done overnight at 16°C)
    J61101


  3. Christine digested minipreps of colonies K, L, M, N, Q, S and T, thought to contain (*a→d*) with PstI. Colonies Q and S showed bands of interest of ~4757bp, ~274bp, and ~1796bp.
  4. Christine set up PCR to test for inserts (*a→d*) and (*d→g*) in TOPO vector. Done with Reddymix and Touch2.
    Primer Combination Gene of Interest Colonies Templates Tested Result
    M13 for + *f for (*d→g*) D M. prep + Colony Non-conclusive
    M13 rev + *f for (*d→g*) D M. prep + Colony Non-conclusive
    M13 for + *b rev (*a→d*) K, L, M, N, Q, S, T M. prep + Colony Non-conclusive
    M13 rev + *b rev (*a→d*) K, L, M, N, Q, S, T M. prep + Colony Non-conclusive
  5. Christine digested TOPO thought to contain (*a→d*) and (*d→g*) with SphI.
    Insert Colony Enzyme Fragments If 5'-3' Fragments If 3'-5' Result
    (*a→d*) K, L, M, N, Q, S, T SphI 2025bp, 4803bp 2595bp, 4232bp
    (*d→g*) D SphI 2843bp, 249bp, 4256bp 3686bp, 3413bp, 249bp

    </ol>

    Thursday 30th August 2007

    Friday 31st August 2007

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