From 2010.igem.org

Lab work

Ordered DNA stocks

The DNA from Mr Gene has finally arrived, now we're ready to build some biobricks!

We were so excited; we immediately dissolved the DNA in water and performed a transformation with 100 ng of the DNA and plated on the LB agar containing the appropriate antibiotic.

AlkB2 (Ampicillin)
rubA3 (Ampicillin)
rubA4 (Ampicillin)
rubB (Kanamycin)
ladA (Ampicillin)
ADH (Ampicillin)
ALDH (Kanamycin)
bbc1 (Ampicillin)
AlnA (Ampicillin)
OprG (Ampicillin)
PalkS1-2 (Ampicillin)
PalkB (Ampicillin)
P(CaiF) (Ampenicillin)
AlkS (Kanamycin)
PhPFD-α (Ampicillin)
PhPFD-β (Ampicillin)

Characterization of Anderson RBS sequences

Yesterday's purified PCR product of I13401 and the Anderson RBS Biobricks (J61100, J61101, J61107, J61117 and J61127) were digested:

#	Sample	Enzyme 1	Enzyme 2	Buffer	BSA	Needed fragment
C1	1.15 μg J61100	SpeI	PstI	2 (BioLabs)	✓	‘S–J61100 pSB1A2–P’
C2	1.68 μg J61101	SpeI	PstI	2 (BioLabs)	✓	‘S–J61101 pSB1A2–P’
C3	1.30 μg J61107	SpeI	PstI	2 (BioLabs)	✓	‘S–J61107 pSB1A2–P’
C4	0.95 μg J61117	SpeI	PstI	2 (BioLabs)	✓	‘S–J61117 pSB1A2–P’
C5	0.51 μg J61127	SpeI	PstI	2 (BioLabs)	✓	‘S–J61127 pSB1A2–P’
D1	2.59 μg I13401	XbaI	PstI	2 (BioLabs)	✓	‘X–I13401–P’
D2	2.65 μg I13401	XbaI	PstI	2 (BioLabs)	✓	‘X–I13401–P’

The digestion products were purified using Roche's PCR Purifiation Kit and loaded onto a 1% agarose gel for comparison with the non-digested BioBricks:

1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 1 μL sample + 4 μL MQ + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane description:

#	Description	Expected Length (bp)	Status
M1	SmartLadder marker (5 μL)	n/a	n/a
1	Undigested J61100 in pSB1A2	2134	✓
3	Undigested J61101 in pSB1A2	2134	✓
4	J61100 + SpeI + PstI	18, 2116	✓
5	J61101 + SpeI + PstI	18, 2116	✓
6	Undigested J61107 in pSB1A2	2134	✓
7	J61107 + SpeI + PstI	18, 2116	✓
8	Undigested J61117 in pSB1A2	2134	✓
9	J61117 + SpeI + PstI	18, 2116	✓
10	Undigested J61127 in pSB1A2	2134	✓
11	J61127 + SpeI + PstI	18, 2116	✓
12	Undigested I13401 in pSB1A2	2936	✓
13	I13401 + XbaI + PstI	883, 2053	✓
14	empty	empty	empty
15	BioRad EZ Load	n/a	n/a

Followed by over night ligation:

#	BioBrick	Recipient plasmid	Fragment	Final volume
1	K398500A	130 μg ‘S–J61100 pSB1A2–P’	154 μg ‘X-I13401-P’	26 μL
2	K398501A	206 μg ‘S–J61101 pSB1A2–P’	247 μg ‘X-I13401-P’	25 μL
3	K398502A	196 μg ‘S–J61107 pSB1A2–P’	247 μg ‘X-I13401-P’	25 μL
4	K398503A	201 μg ‘S–J61117 pSB1A2–P’	247 μg ‘X-I13401-P’	25 μL
5	K398504A	202 μg ‘S–J61127 pSB1A2–P’	247 μg ‘X-I13401-P’	25.5 μL
6	negative control	123 μg ‘S–J61117 pSB1A2–P’	-	15 μL

To all samples one-tenth of the total volume ligase buffer was added.

Emulsifier

Pieter is testing different condition for the emulsifying Assay. To determine the emulsifying activity we set up a calibration curve with SDS. He prepared the following samples: