From 2007.igem.org

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Prepared for site dirrected mutagenesis

• Diluted DNA from last round to give 10ng/37ul

Tube	conc of miniprep ng/ul total=(100ul)	x uL miniprep added to y mL milliQ
21A	192	2ul in 1.42ml
22A	176	2ul in 1.3ml
23A	247	1ul in 0.91ml

Site dirrected Mutagenesis Round #3

• 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.

• PCR conditions:
  • seg 1 95 degC 2min x 1
  • seg 2 x 18
    • 95 deg 1min
    • 60 deg 50 sec
    • 68 deg 15min
  • seg 3 68 deg 7min x 1
  • seg 4 4 deg indefinately

Site dirrected Mutagenesis round #3

Mutation Number	Template DNA (10ng)	Sence Primer	Antisence Primer	#Colonies	Picks named
31	21A	GvpL-g696a	GvpL-g696a-R	lots	31A,31B
32	22A	GvpL-g213a	GvpL-g213a-R	lots	32A,32B
33	23A	GvpQ-g150a	GvpQ-g150a-R	lots	33A,33B

• Picked colonies from Plates and culture overnight
  

  

  PstI digest
• 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
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)->	31	117	133
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)->	32	107	96
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)->	33	114	152

• Diagnostic digests were performed using
  • A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 1h30min. (25uL reaction)

• Prepared two 8 lane 50mL agarose gel
• Loaded 20uL of digest
• Digest Pattern
• Expected effects

PstI digest

History	Mutation tube	FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)->	31		5983	2999	<-	-
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)->	32		6466	2516	<-	-
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)->	33		7318	<-	2516	378	-	105

• Results of PstI diagnostics

Conclude 32B and 33A are good, and 31A & B are disturbingly different but similar so we should take both of them forward. Poor Jell though.