Team:Groningen/Labwork/16 July 2013

Mirjam

Finally the colonies are growing on LB agar plates with ampicillin. The negative control does not show any colonies, the double negative does??, the positive control shows a lot of red colonies. On one of the four plates there are no colonies present, on two of them only a couple and on one a lot. The colonies are picked and placed in LB medium with ampicillin to grow further for miniprep. Colony PCR revealed no bands. Mini prep analysis is done with the colonies of plate 1 and 3.
restriction analysis is done with EcoRV. Picked colonies from the plate with the backbone of Munich (BBa_K823023) for overnight inoculation in LB medium with ampicillin. To obtain more sample of this backbone.

Inne

Did a PCR with 3 samples in duplo

Sample	Primer F	Primer R	Annealing temp.
1	without strep tag	without strep tag	78 C
2	with strep tag	without strep tag	78 C
3	without strep tag	with strep tag	80 C

Protocol:

amount	compound
28.2 uL	MilliQ
1.5 uL	5% DMSO
1 uL	each dNTP
10 uL	Phusion GC buffer
2.5 uL	Primer F
2.5 uL	Primer R
1 uL	Silk template BBa_16
0.3 uL	Phusion polymerase

Ran a gel 1.5% agarose using the big slots,
gel ran at 90 V for 45 minutes.
gel didn't run nice, tried to cut out bands from 2 and 3.
did gel purification
Ran 0.8% agarose gel for 24 min on 100 V
Turned out that the bands were a bit higher then 1kb
made new TBE buffer. 100ml TBE/900ml demiwater.
made the inoculation of the E.coli with the BBa_k818000 biobrick for the Delft iGEM team.
Cells inoculated in 4 ml LB liquid medium with 8 uL of stock Ampicillin
Cells are to grow overnight in a 37 C shaker.

Sander

did a restriction digestion for Silk and Signal Sequences.

Sample	componant	volume (ul)
silk1	silk	4
	BamHI buffer	2
	BamHI enzyme	0,5
silk2	silk	4
	BamHI buffer	2
	BamHI enzyme	0,5
silk3	silk	4
	BamHI buffer	2
	BamHI enzyme	0,5
SS MotB	MotB	2
	BamHI buffer	2
	BamHI enzyme	0,5
SS FliZ	Fliz	2
	BamHI buffer	2
	BamHI enzyme	0,5
SS EstA	EstA	2
	BamHI buffer	2
	BamHI enzyme	0,5
SS LytB	LytB	2
	BamHI buffer	2
	BamHI enzyme	0,5

MilliQ was added to reach a 20 ul volume.
Incubation started at 11:00 for the signal sequences and 11:30 for the silk.
at 15:38 the restriction digestion reaction was run over a 1,5 % agarose gel with 100 v for 22 min.
no result on the gel for the sik. results as expected for signal sequence.

Claudio and Mike

The synthetic genes design goes on.
Today we focus on codon optimizing the sequences for Bacillus Subtilis taking into account the codon usage.