Team:Freiburg/Notebook/5 September

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green light receptor

transformation with reporter parts

Investigators:Julia

transformation with iGEM parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15016 BBa_I15016] which is B0032.ECFP
and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15017 BBa_I15017 ]which is B0032.EYFP.
Tomorrow the green light promotor from PCR of 29_August should be inserted infront of the reporter proteins CFP and YFP.

blue light receptor

Miniprep

Investigators:Sophie
continue from: picking clones; 04.09.11

Testdigest showed no inserts

Ligation

Investigators: Sophie
again ligation as last time (02.08.11)

Lysis cassette

Troubleshooting of the modified Lysis genes K124017

Investigators:Theo

Minipreps of the first inoculations were applied on a 1% agarose gel without digesting them to see if they have an insert of the modified Lysis genes K124017. Nr1, 5 and 7 were sent for sequencing since the size seems to be about right.

Note: S4 is the B0034 RBS (ribosome binding site), S15 is the non-modified K124017 Lysis Cassette without RBS

Precipitator

Ligation

Name:Rüdiger	Date: 05.09.
Continue from Digestion Date 05.09 Name Rüdiger Experiment
Project Name: Precipitator

Procedure

PCR tube:

total volume 20 μl

add H₂O (17 μl -X-Y-Z)
add 2 μl Ligase Buffer 10x
add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
Add 1 μl T4-DNA Ligase
Incubate 10-30 min at room temperature
heat for 20 minutes at 80°C
store at -20°C or directly proceed to transformation

	Name of part	Ratio Insert:Vector = 3:1 or 1:1	Volume (μl)
X insert 1
Y insert 2
Z vector
H₂O

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.

All samples from last digestion.

Name	Vector	Insert
1a	PGEX 1-3V	1
2a	PGEX 1-3V	2
3a	PGEX 1-3V	3
4a	C3	4
5a	PET DUET	5
6a	PET DUET	6
7a	PET DUET	7
8a	C3	8
9a	C3	9
10a	C3	10
1b	pGEX	1
2b	pGEX	2
3b	pGEX	3
4b	pGEX	4
5b	PET DUET	5
6b	PET DUET	6
7b	PET DUET	7
8b	C3	8
9b	C3	9
10b	C3	10

Digestion

Name:Rüdiger	Date: 05.09.
Continue from Digestion Date 31.08. Name Rüdiger Experiment
Project Name: Precipitator

Procedure

add H₂O (38μl-DNA )
5 μl NEB4 buffer (stored at iGEM’s, -20°C)
5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
1 μl restriction enzymes (stored at iGEM’s, -20°C)
heat for 1-2 hours 37°C (6 hours if time)
heat for 20 minutes 80°C (inactivation of enzymes)
keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Sample Name	DNA concentration (μg/μl)

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Name	Vector (+Antarctic phosphatase 1 μl +AP buffer 5,6 μl)	Insert (+Dpn1 1 μl)	Enzyme1	Enzyme2
1	PGEX David	30	EcoR1	Bam H1
2	PGEX David	30	EcoR1	Bam H1
3	PGEX David	30	EcoR1	Bam H1
4	C3	8	EcoR1	Spc1
5	PET DUET	12	EcoR1	Pst1
6	PET DUET	22	EcoR1	Pst1
7	PET DUET	20	EcoR1	Pst1
8	C3	20	EcoR1	Spc1
9	C3	20	EcoR1	Spc1
10	C3	15	EcoR1	Spc1