Glasgow/Wetlab/Week9

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Latest revision as of 12:34, 25 October 2007

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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 phzD→phzG 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 phzA→phzD, from plate 2.

Tuesday 28 August 2007

1. Christine, Lynsey and Maia 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 of interest of ~6.7kb.
2. Christine digested all minipreps of interest for phzA→phzD and miniprep D of phzD→phzG with EcoRI. However, none showed bands of interest - possibly a fault of the enzyme, new EcoRI ordered.

   Insert	phzA→phzD	phzD→phzG
   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 phzA→phzD and phzD→phzG in TOPO vector. Done with Reddymix and Touch2.

   Primer Combination	Gene of Interest	Colonies	Templates Tested	Result
   M13 for + *f for	phzD→phzG	D	M. prep + Colony	Non-conclusive
   M13 rev + *f for	phzD→phzG	D	M. prep + Colony	Non-conclusive
   M13 for + *b rev	phzA→phzD	K, L, M, N, Q, S, T	M. prep + Colony	Non-conclusive
   M13 rev + *b rev	phzA→phzD	K, L, M, N, Q, S, T	M. prep + Colony	Non-conclusive

5. Christine digested TOPO thought to contain phzA→phzD and phzD→phzG with SphI.

   Insert	Colony	Enzyme	Fragments If 5'-3'	Fragments If 3'-5'	Result
   phzA→phzD	K, L, M, N, Q, S, T	SphI	2025bp, 4803bp	2595bp, 4232bp
   phzD→phzG	D	SphI	2843bp, 249bp, 4256bp	3686bp, 3413bp, 249bp

6. Items sent off for sequencing: phzA→phzD colonies Q and S, phzD→phzG colony D.

   Thursday 30th August 2007

   1. Christine began site directed mutagenesis of phzA→phzD Q and S, and phzD→phzG D by setting up the following PCR with KOD and SDM_KOD Program with extension time of 7 min 30 secs. Labelled as follows:
      This table is incorrect, see 04/09/07.

      PCR tube	Colony DNA is taken from	Insert	Primers used
      1A	D	phzA→phzD	EcoRI *B for + EcoRI *B rev
      1B	D	phzA→phzD	PstI *C for + PstI *C rev
      1C	S	phzD→phzG	EcoRI *E for + EcoRI *E rev
      1D	S	phzD→phzG	PstI *E for + PstI *E rev
      1E	S	phzD→phzG	PstI *F for + PstI *F rev
      1F	Q	phzD→phzG	EcoRI *E for + EcoRI *E rev
      1G	Q	phzD→phzG	PstI *E for + PstI *E rev
      1H	Q	phzD→phzG	PstI *F for + PstI *F rev

   Friday 31st August 2007

   1. Christine began site directed mutagenesis of TOPO containing phzA→phzD and phzD→phzG by digesting PCR of colonies D, Q and S (see 30/08/07) with DpnI (see Protocol), and transforming them into TOP10 cells and plating them on carbenicillin plates.

   
   

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