Glasgow/Wetlab/Week9
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Week 9
Monday 27th August 2007
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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 - 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.
- 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
- 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.
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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
- 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...
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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 - 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.
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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 -
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 -
Items sent off for sequencing: phzA→phzD colonies Q and S, phzD→phzG colony D.
Thursday 30th August 2007
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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
- 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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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:
