From 2007.igem.org

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Prepared for site dirrected mutagenesis

• Produced NZY broth 100mL autoclaved (due to contamination in last batch).
• Diluted DNA from last round

Tube	conc of miniprep ng/ul total=(100ul)	1uL miniprep added to x uL milliQ
5A	141	522
6A	212	784
11A	221	817
10B	253	936

Site dirrected Mutagenesis Round #2

• 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.

Changed PCR conditions on some primers that didn't work before to see if they would work. Specifically segment1 increased from 1 minute @ 95deg to 2minutes, segmet 2 increased from 50 sec @ 95 deg to 1 minute, and elongation time increased from 10 min @ 68 deg to 15 minutes.

Site dirrected Mutagenesis round #2

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.
  • 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 2uL of miniprep(100uL) diluted in 98uL of TE.

DNA concentrations in ng/uL

History	Mutation tube\colony:	A	B	C	D	E
pNL29T1-(GvpP-g441a)->	3	141	112
pNL29T1-(GvpL-g318a)->	4	108	218
pNL29T1-(Comp-g318a)->	5	141	119
pNL29T1-(GvpL-g351a)->	6	212
pNL26P3-(GvpQ-g183a)->	10	305	253	258	392	272
pNL26P3-(GvpP-g441a)->	11	221	190	259	204

• 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	pNL29T1		5983	2516	378	105
pNL29T1-(GvpP-g441a)->	3		6088	2516	378	<-
pNL29T1-(GvpL-g318a)->	4		5983	2516	483	<-
pNL29T1-(Comp-g318a)->	5		5983	2516	483	<-
pNL29T1-(GvpL-g351a)->	6		5983	2516	378	105
pNL26P3	pNL26P3		3928	3137	2516	378	220	105	33
p26P3-(GvpQ-g183a)->	10		3928	3170	2516	378	220	105	<-
p26P3-(GvpP-g441a)->	11		4148	3137	2516	378	<-	105	33

EcoRI and BamHI digest

History	Mutation tube	Fragments:
pNL29T1	pNL29T1		6124	2112	747
pNL29T1-(GvpP-g441a)->	3		6124	2112	747
pNL29T1-(GvpL-g318a)->	4		6124	2112	747
pNL29T1-(Comp-g318a)->	5		6124	2112	747
pNL29T1-(GvpL-g351a)->	6		6871	2112	<-

• Results of PstI diagnostics:
  

  

  PstI digest
  • pNL29 control was barely visable.
  • Bands less than 300bp were not visable
  • Colonies 5A,5B,5C,and 3C all showed bands at 483bp and not at 378bp as required although 5B and 3C appear slightly larger than the other two.
  • Colony 6A shows no band at 747bp and a band at 6871bp as required.
  • Colonies 4A,and 4B contain a band at 378bp indicating failure of mutagenesis.
  • The following colonies show additional bands of unknown origin: 3B and 4C (700bp) , 3A (8500bp) of unknown origin.
  • See Gel
  • Conclude that 5A and 6A are suitable templates for the second round of mutagenesis.
• Results of EcoRI diagnostics:
  

  

  EcoRI & BamHI digests
  • pNL26 control barely visable.
  • Colonies 11A,B,C,D and 10A,B,C,D,E all show required pattern but the 33bp band is unlikely to be visible making the 10A..E test inconclusive, and the 220bp band in 11A..D is only barely visible in 11A..E making its diagnosis also risky.
  • See Gel
  • Conclude that 10B and 11A may be suitable templates for the second round of mutagenesis.
  • Conclude that Primers generated by the stratagene program Comp-g318a outperformed (60 colonies of which 2/3 provided correct product ) both offset primers JMY-g318a (26 colonies of which 2/3 gave template DNA)primers and hand designed primers Gvp-g318a (3 colonies of which when using the stratagene PCR protocol.

Sucess of primers gnerated different ways:

Primers name	Design protocol	no colonies	#correctly mutated	#unknown	# template
(Comp-g318a)	Statagene	60	2	1	-
(JMY-g318a)	Offset	26	-	1	2
(GvpL-g318a)	Hand design	3	-	3	-

• It was however also noted that the success of the PCR with the hand generated primers was negatively correlated with the melting temperature of the primers.
• It was therefore decided to increase the initial melting time from 1 to 2 minutes and increase subsequent melting times from 50 seconds to 1 minute in the next round of mutagenesis.