Glasgow/Wetlab/Week10

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Week 10

Monday 3rd September 2007

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.

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

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	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

</ol>

Friday 7th September 2007

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 === Tuesday 4th September 2007 ===

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