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Revision as of 01:58, 26 October 2007

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Prepared for site dirrected mutagenesis

• Diluted DNA from last round, creating 10ng/ul sample and then adding 1uL of this to 36uL milliQ water to produce required 10ng/37uL template for PCR.

Tube	conc of miniprep ng/ul total=(100ul)	1uL miniprep added to x uL milliQ -> 10ng/uL
31A	117	10.7
31B	133	12.3
32B	96	8.6
33A	114	10.4

Site dirrected Mutagenesis Round #4

• Applied the stratagene Site directed mutagenesis protocolto the following DNA and primer pairs, which when plated out on LB AMP plates producing the numbers of colonies shown. Several of these were picked and tubes marked with a character suffix as shown in table.

Site dirrected Mutagenesis round #4

Mutation Number	Template DNA (10ng)	Sence Primer	Antisence Primer	#Colonies	Picks named
21	5A	GvpL-g351a	GvpL-g351a-R	21A,21B
22	6A	GvpL-g318a	GvpL-g318a-R	3	22A,22B,22C
23	11A	GvpQ-g183a	GvpQ-g183a-R	26	23A,23B
24	10B	GvpP-g441a	GvpP-g441a-R	60	24A,24B
25	6A	GvpL-g696a	GvpL-g696a-R	1	25A,25B (new PCR conditions)
26	6A	GvpL-g213a	GvpL-g213a-R		26A,26B (new PCR conditions)
27	10B	GvpQ-g150a	GvpQ-g150a-R		27A,27B (new PCR conditions)
28	10B	GvpQ-g150a	GvpQ-g150a-R	None

• Ran gel of 10uL sample of pcr reaction with 1uL of bluejuice.
  

  

  PCR product run on gel
  • showed lane GvpQ-g150a non functional in old conditions but functional in new conditions.
• Picked colonies from Plates and culture overnight
• Produced glycerol stocks from 900uL of each overnight culture.
• Minipreped remaining culture (4mL) giving 100uL.
• Measure DNA concentrations using 5uL of miniprep(100uL) diluted in 45uL of TE.

DNA concentrations in ng/uL

History	Mutation tube\colony:	A	B	C	D	E
pNL29T1-(Comp-g318a)-(GvpL-g351a)->	21	183
pNL29T1-(GvpL-g351a)-(GvpL-g318a)->	22	176
pNL26P3-(GvpP-g441a)-(GvpQ-g183a)->	23	247
pNL26P3-(GvpQ-g183a)-(GvpP-g441a)->	24
pNL29T1-(GvpL-g351a)-(GvpL-g696a)->	25
pNL29T1-(GvpL-g351a)-(GvpL-g213a)->	26
pNL26P3-(GvpQ-g183a)-(GvpQ-g150a)->	27

• Diagnostic digests were performed using
  • A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 2h30min. (25uL reaction)
  • B) 21.5uL of each p29T1 based colony in Buffer2 with 1uL EcoRI (20U) and 1uL BamHI (20U) at 37degC for 2h30min. (26uL reaction)
  • C) 21.5uL of each pNL26P3 based colony and in Buffer2 with 1uL EcoRI (20U) at 37degC for 2h30min. (25uL reaction)
• Prepared two 20 lane 100mL agarose gel
• Loaded 20uL of digest
• Digest Pattern
• Expected effects

PstI digest

History	Mutation tube	FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a)->	21		5983	2516	483	<-
pNL29T1-(GvpL-g351a)-(GvpL-g318a)->	22		5983	2516	483	<-
pNL26P3-(GvpP-g441a)-(GvpQ-g183a)->	23		4148	3170	2516	378	<-	105
pNL26P3-(GvpQ-g183a)-(GvpP-g441a)->	24		4148	3170	2516	378	<-	105
pNL29T1-(GvpL-g351a)-(GvpL-g696a)->	25		5983	2894	<-	105
pNL29T1-(GvpL-g351a)-(GvpL-g213a)->	26		6088	2516	378	<-
pNL26P3-(GvpQ-g183a)-(GvpQ-g150a)->	27		3928	3390	2516	378	<-	105

• Results of PstI diagnostics:
  

  

  PstI digest

EcoRI and BamHI digest

History	Mutation tube	Fragments:
pNL29T1-(Comp-g318a)-(GvpL-g351a)->	21		6871	2112	<-
pNL29T1-(GvpL-g351a)-(GvpL-g318a)->	22		6871	2112	<-
pNL29T1-(GvpL-g351a)-(GvpL-g696a)->	25		6871	2112	<-
pNL29T1-(GvpL-g351a)-(GvpL-g213a)->	26		6871	2112	<-

• Results of EcoRI diagnostics:

Conclude 21A,22A,23A are all satisfactory.