Team:Freiburg/Notebook/14 July

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red light receptor

3A-assembly: Digest

Investigators: Jakob

3A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)

Digestion

Name:Jakob	Date: 14.07.2011
Project Name: 3A-assembly of pcyA+ terminator, ho1+terminator

Procedure

1. add H2O (16μl-DNA )

2. 2μl NEB4 buffer

3. 2 μl 10x BSA (used 1:10 diluted )

4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)

5. 1 μl restriction enzymes

6. heat for 1-2 hours 37°C (6 hours if time)

7. heat for 20 minutes 80°C (inactivation of enzymes)

8. 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)
Vector pSB1T3	20
terminator	38.8
ho1	176
pcyA	173

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

Components	Vector (μl)	Insert1 and 2 (μl)
DNA (μl)	2	Ho1 2.5 pcyA 2.5	Terminator 5.5 terminator 5.5
BSA (10x) (2μl)
NEB4 Buffer (2μl)
Enzyme 1 (1μl)	EcoRI	EcoRI	XbaI
Enzyme 2 (1μl)	PstI	SpeI	PstI
H2O (14 μl- DNA)
In total 20 μl

3A-assembly: Ligation and transformation

Investigators: Julia

Ligation of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator) in vector pSB1T3.

L23: Ligation of S23+S21a in pSB1T3 L22: Ligation of S22+S21a in pSB1T3 Ligation

Name:Julia	Date:14.07
Continue from Date14.07 Name Experiment 3A-assembly
Project Name:3A-assembly of pcyA+ terminator, ho1+terminator

Procedure

total volume 20 μl

1. add H2O(17 μl -X-Y-Z)

2. add 2 μl Ligase Buffer 10x

3. add Insert 1, Insert 2 (when proceeding from 3A digestion use 2 μl of each)

4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)

5. Add 1 μl T4-DNA Ligase

6. Incubate 10-30 min at room temperature

7. heat for 20 minutes at 80°C

8. store at -20°C or directly proceed to transformation

	Name of part	Ratio Insert:Vector 1:1	Volume (μl)
X insert 1	ho1/ pcyA		2
Y insert 2	terminator		2
Z vector	pSB1C3		2.5
H2O			11

Competent neb10 strain was transformed with 2μl ligation reaction and plated out.

PCR

Investigators: Julia

PCR of the red light receptor, cph8.

Primer:

P10	atatgaattcgcggccgcttctagATGGCCACCACCGTAC
P11	atatatgcatctgcagcggccgctactagtaTTACCCTTCTTTTGTCaTGCC

DNA template:

BBa_I150010

Programm: 2min 55/60

Lysis cassette

Quickchange PCR

Name: Theo

Date: 14.07.2011

Continue from Experiment Lysis Cassette

Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from

Lysis Device

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H₂0	Name
10µl	6x HF Phusion Buffer
2.5µl	Primer fw	P29 (1:10)
2.5µl	Primer dw	P30 (1:10)
1µl	dNTPs
1µl	DNA-Template	M15a (from S15->diluted to 5ng/µl)
0.5 µl	Phusion (add in the end)

What program do you use?

	95°C - 15 sec
95°C - 5 min	55°C - 15 sec	72°C -5 min	16°C - ∞
	72°C - 5’ + 5’’
	20x

Precipitator

Test-digest

Investigators: Rüdiger

Testdigest of the minipreps, Sophie did (june, 13).

Enzyme 1: PstI
Enzyme 2: EcoRI

Image of different samples from miniprep.