Glasgow/Wetlab/Week10

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Revision as of 21:12, 2 October 2007

Week 10

Monday 3rd September 2007

1. 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 miniprepped 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

1. Christine realised that the site directed mutagenesis she had attempted on Thursday 30/08/07 was mistaken. 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.
2. 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.

3. 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

1. 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

2. 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

1. 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	Original Colony	Insert	Site Mutated	Enzyme	Expected Bands	Result
   1A	D	(*e→g*)	EcoRI (679)	EcoRI	7046bp, 18bp, 284bp	No bands of interest
   1B	D	(*e→g*)	PstI (1688)	PstI	2511bp, 3682bp, 1155bp	No bands of interest
   1C	D	(*e→g*)	PstI (2237)	PstI	1704bp, 1962bp, 3682bp	No bands of interest

Friday 7th September 2007