Glasgow/Wetlab/Week10
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Week 10
Monday 3rd September 2007
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Christine's plates from 31/08/07 all grew except for 1B. Overnights were set up from 7 colonies on each of the plates on Sunday, labelled 1A1→1H7, 49 in total as there were none from plate 1B. Christine, Maia and Maija miniprpped them and digested them as follows:
This table is incorrect, see 04/09/07.PCR tube Colony DNA is taken from Insert Enzyme Expected sizes Result 1A D (*a→d*) EcoRI 3455bp, 18bp, 3654bp Negative 1B D (*a→d*) PstI 274bp, 6553bp Negative 1C S (*d→g*) EcoRI 7046bp, 18bp, 284bp Negative 1D S (*d→g*) PstI 1155bp, 2511bp, 3682bp Negative 1E S (*d→g*) PstI 1962bp, 1704bp, 3682bp Negative 1F Q (*d→g*) EcoRI 7046bp, 18bp, 284bp Negative 1G Q (*d→g*) PstI 1155bp, 2511bp, 3682bp Negative 1H Q (*d→g*) PstI 1962bp, 1704bp, 3682bp Negative Tuesday 4th September 2007
- Christine realised that the site directed mutagenesis she had attempted on Thursday 30/08/07 was wrong. S and Q were meant to be tested for (*a→d*), and D was meant to be tested for (*d→g*) but S and Q were tested for (*d→g*), and D was tested for (*a→d*). This means the digests from 03/09/07 were also incorrect. However, all is not lost because the sequencing results for S and D came back today and they show that both S and D contain (*d→g*). This means that, theoretically, digests of mutations 1C, 1D and 1E from 03/09/07 should be positive, but this is not the case.
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Before the sequencing results for S and D arrived, Christine set up site directed mutagenesis to correct that which she did 30/08/07. Used KOD and KOD_SDM programme as 30/08/07.
PCR tube Colony DNA is taken from Insert Primers used 1A D (*e→g*) EcoRI *E for + EcoRI *E rev 1B D (*e→g*) PstI *E for + PstI *E rev 1C D (*e→g*) PstI *F for + PstI *F rev 1D S (*a→d*) EcoRI *B for + EcoRI *B rev 1E S (*a→d*) PstI *C for + PstI *C rev 1F Q (*a→d*) EcoRI *B for + EcoRI *B rev 1G Q (*a→d*) PstI *C for + PstI *C rev After the PCR raction, these were each digested with DpnI for 1 hour at 37°C and PCR purified according to QIAGEN manual. After this they were transformed (15ul DNA into half a tube of TOP10 cells, heat shock of 30 seconds at 42°C, plated on carb) and labelled alphabetically according to PCR tubes above.
- Maija set up overnights in LB carb of colonies thought to contain (*a→d*) which were plated 23/08/07, labelled 1→10.
Wednesday 5th September 2007
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Maia, Maija and Christine miniprepped Maija's overnights of colonies thought to contain (*a→d*). They were then digested with EcoRI and PstI.
Insert Enzyme Expected Sizes(5'→3') Expected Sizes (3'→5') Result (*a→d*) EcoRI 928bp, 2227bp, 18bp, 3654bp 1796bp, 1349bp, 3682bp No bands of interest (*a→d*) PstI 4757bp, 274bp, 1796bp 1796bp, 1349bp, 3682bp No bands of interest - Christine set up overnights from yesterday's site directed mutagenesis transformations, 7 colonies per plate were seeded, labelled A, B and C according to PCR tube (D, E, F, and G did not contain what they were originally thought to, see 04/09/07), and each colony labelled 1→7.
Thursday 6th September 2007
Lynsey, Maia and Maija miniprepped Chrisitne's overnights of colonies containing (*e→g*) which had undergone one round of site directed mutagenesis. They were the digested as follows:
Label Insert Site Mutated Enzyme Expected Bands 1A (*e→g*) EcoRI (679) EcoRI 7046bp, 18bp, 284bp 1B (*e→g*) PstI (1688) PstI 2511bp, 3682bp, 1155bp 1C (*e→g*) PstI (2237) PstI 1704bp, 1962bp, 3682bp Friday 7th September 2007
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