Glasgow/Wetlab/Week9

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Revision as of 12:30, 8 October 2007

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. 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 (*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

6. Items sent off for sequencing: (*a→d*) colonies Q and S, (*d→g*) colony D. </li>

   Thursday 30th August 2007

   1. Christine began site directed mutagenesis of (*a→d*)Q and S, and (*d→g*) 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	(*a→d*)	EcoRI *B for + EcoRI *B rev
   1B	D	(*a→d*)	PstI *C for + PstI *C rev
   1C	S	(*d→g*)	EcoRI *E for + EcoRI *E rev
   1D	S	(*d→g*)	PstI *E for + PstI *E rev
   1E	S	(*d→g*)	PstI *F for + PstI *F rev
   1F	Q	(*d→g*)	EcoRI *E for + EcoRI *E rev
   1G	Q	(*d→g*)	PstI *E for + PstI *E rev
   1H	Q	(*d→g*)	PstI *F for + PstI *F rev

   Friday 31st August 2007

   1. Christine began site directed mutagenesis of TOPO containing (*a→d*) and (*d→g*) 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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