Team:Heidelberg/Notebook/material

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Revision as of 17:46, 28 October 2008

Material

Buffers & Solution


SM	50 mM	Tris, pH 7.5
	20 mM	MgSO4
	50 mM	NaCl
	0.01 %	gelatine

Tethering buffer, pH 7.0	10 mM	potassium phosphate
		(K2HPO4 : KH2PO4 = 1 :1 )
	100 µM	EDTA
	1 µM	L-Methionine
	10 mM	lactic acid

M63 salt	3 g/l	KH2PO4
	9 g/l	K2HPO4
	4 g/l	(NH4)2SO4
	0.5 g/l	FeSO4

Amino acid mix	5 g/l	L-threonine
	5 g/l	L-histidin
	5 g/l	L-leucine
	5 g/l	L-methionine

M9 salts	64 g/l	Na2HPO4 x 7H2O
	15 g/l	KH2PO4
	2.5 g/l	NaCl
	5 g/l	NH4Cl

Kits

Kit	supplier

CompactPrep Plasmid Maxi Kit	Qiagen
HISpeed Plasmid Maxi Kit	Qiagen
MaxPlax™ Lambda Packaging Extracts	EPICENTRE Biotechnologies
QIAEX II Gel Extraction Kit	Qiagen
QIAGEN Lambda Mini Kit	Qiagen
QIAprep Spin Mini Kit	Qiagen
QIAquick Gel Extraction Kit	Qiagen
QIAquick PCR Purification Kit	Qiagen

Marker

Marker	supplier

GeneRuler™ High Range DNA Ladder	MBI Fermentas
GeneRuler™ 1kb DNA Ladder Mix	MBI Fermentas
GeneRuler™ 1kb Plus DNA Ladder Mix	MBI Fermentas

Enzymes

Enzym	supplier

Pfu DNA polymerase	Stratagene
Pfu turbo DNA polymerase	Stratagene
Phusion DNA polymerase	Finnzymes
Taq DNA polymerase	MBI Fermentas
T4 DNA ligase	MBI Fermentas / New England Biolabs

AgeI	New England Biolabs
BamHI	New England Biolabs
BglI	MBI Fermentas
BseJI	MBI Fermentas
BspEI	New England Biolabs
DpnI	Roche Diagnostics / New England Biolabs
DraI	New England Biolabs
EcoRI	New England Biolabs
EcoRV	New England Biolabs
HindIII	New England Biolabs
KpnI	New England Biolabs
NcoI	New England Biolabs
NdeI	New England Biolabs
NotI	New England Biolabs
PstI	New England Biolabs
SacI	New England Biolabs
SalI	MBI Fermentas
ScaI	New England Biolabs
SfcI	New England Biolabs
SmiI	MBI Fermentas
SpeI	New England Biolabs
SpeI	New England Biolabs
XbaI	New England Biolabs
XhoI	MBI Fermentas
XmaI	New England Biolabs

Plasmidvectors

Name	application	reference

BBa_B0015	terminator	http://partsregistry.org
BBa_F1610	LuxI	http://partsregistry.org
BBa_I15030	AI-1 amplifier	http://partsregistry.org
BBa_I20260	GFP	http://partsregistry.org
BBa_J01003	oriT	http://partsregistry.org
BBa_J16002	cloning vector	http://partsregistry.org
BBa_J23107	constitutive promotor	http://partsregistry.org
BBa_T9002	AI-1 GFP receiver	http://partsregistry.org
CheY-mCherry	cloning vector	V. Sourjik, ZMBH
ColE1	colicin E1	DSMZ
ColE9-J	colicin E9	C. Kleanthous, University of York
pBad18	cloning vector	V. Sourjik, ZMBH
pBad33	cloning vector	V. Sourjik, ZMBH
pBluescript II SK (+)	cloning vector	V. Sourjik, ZMBH
pDest15	cloning vector	DKFZ Library
pDK4	visualization	V. Sourjik, ZMBH
pDK48	cloning vector	V. Sourjik, ZMBH
pDK58	cloning vector	V. Sourjik, ZMBH
pDK6	visualization	V. Sourjik, ZMBH
pED374	oriT	K. Derbyshire, Wadsworth
pES16	cloning vector	V. Sourjik, ZMBH
pMMB863	oriT	M. Bagdasarian, MSU
pQE-30	cloning vector	Invitrogen
pSB1A2	cloning vector	http://partsregistry.org
pSB2K3	cloning vector	http://partsregistry.org
pTrc99a	cloning vector	V. Sourjik, ZMBH
pUB307	helper plasmid	E. Lanka, BfR
RP4	helper plasmid	M. Bagdasarian, MSU

Synthetic oligonucleotides

All oligonucleotides were purchased from Invitrogen (Karlsruhe) and adjusted to a standard concentration of 100 ^pmol/_µl.

Name	SEQUENCE (5´->3´)

Bam_fw	GACAAGTGTTGGCCATGGAACAGG
Bam_rv	GCCGTCTGTGATGGCTTCCATG
cI_mut_fw	GCGTCTGGGTGGTGATGAGTTCACCTTCAAAAAACTG
cI_mut_rv	CAGTTTTTTGAAGGTGAACTCATCACCACCCAGACGC
CmR_EcoRI_mut_fw	GAATGCTCATCCGGAGTTCCGTATGGCAATG
CmR_EcoRI_mut_rev	CATTGCCATACGGAACTCCGGATGAGCATTC
CmR_fw	GCTAAAATGGAGAAAAAAATCACTGG
CmR_new_fw	TACGAGGTACCTTTACAGCTAGCTCAGTCCTAGGTATTATGC
CmR_new_rev	TATATAAGCTTTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTG
CmR_Prefix_fw	GAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGG
CmR_rv	AGGTTCTCCTTTATTAGCCGGATCCTCTAGATTACGCC
CmR_Suffix_rv	CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGCCCACCC
colE1_kil_prot_rv_A_SpeI	TATATACTAGTACTACTGAACCGCGATCCCCG
colE1_mut_EcoRI_fw	GGTATTGCTATTGTTACAGGTATTCTATGCTCCTATATTGATAAG
colE1_mut_EcoRI_rv	CTTATCAATATAGGAGCATAGAATACCTGTAACAATAGCAATACC
colE1_mut_PstI_1_fw	GCAGTAAAAGTGAAAGTTCAGCAGCTATTCATGCAACTGC
colE1_mut_PstI_1_rv	GCAGTTGCATGAATAGCTGCTGAACTTTCACTTTTACTGC
colE1_mut_PstI_2_fw	GCTGCCCGGGCAAAAGCAGCAGCGGAAGCACAGG
colE1_mut_PstI_2_rv	CCTGTGCTTCCGCTGCTGCTTTTGCCCGGGCAGC
colE1_mut_PstI_3_fw	CATTAGAGAAGAAAGCAGCAGATGCAGGGGTGAG
colE1_mut_PstI_3_rv	CTCACCCCTGCATCTGCTGCTTTCTTCTCTAATG
colE1_prot_fw_BamH1	TACGAGGATCCATGGAAACCGCGGTAGCG
colE1_prot_rv_HindIII	TATATAAGCTTTTAAATCCCTAACACCTC
colE9_lysProt_rv_A_SpeI	TATATACTAGTACTAGGTTTTCGGCTTAGAACCCC
colE9_mut_EcoRI_fw	GTTGGGTGGACGATTCGAGAGTTCAATGGGGAAATAAAAATG
colE9_mut_EcoRI_rv	CATTTTTATTTCCCCATTGAACTCTCGAATCGTCCACCCAAC
colE9_plasmid_rv_A_SpeI	TATATACTAGTACACATGGAACTTTTGTGAC
colE9_prot_fw_BamH1	TACGAGGATCCATCGATTTGCCCATGACCC
colE9_prot_rv_XmaI	TATATCCCGGGTTACTTACCTCGGTGAATATCG
DK13	TCACCCGCACGCGC
DK167	AGGATACTAGTAGGCCATTACTTT
DK9b	CGATGCGGCCGCTCAAAATGTTTCCCAGTTTGG
F1610_fw_XbaI	TACGATCTAGAAAAGAGGAGAAATACTAG
F1610_rv_HindIII	TATATAAGCTTTATAAACGCAGAAAGGCCC
GAM_fw	AGTGCTTTAGCGTTAACTTCCG
GAM_rv	GGTTTTACCGCATACCAATAACG
GFP_CmR_fw	CTCGTTGGTACCTCTAGATTTACAGCTAGCTCAGTCCTAGG
GFP_CmR_rv	TATTCGACCGGTACTAGTTATAAACGCAGAAAGGCCCACC
GFP_new_fw	TACGAGAGCTCTTTACAGCTAGCTCAGTCCTAGG
GFP_new_rv	TATATACCGGTACTAGTTATAAACGCAGAAAGGCCCACC
Lambda_insert_fw	TTGTAAAAACAGCCCTCCTC
Lambda_insert_rv	GATATGACTATCAAGGCCGC
LuxP_mut_F	CGTGAATTAGCAACAGAGTTCGGAAAGTTCTTCCC
LuxP_mut_R	GGGAAGAACTTTCCGAACTCTGTTGCTAATTCACG
LuxP_prefix_F	GAGGGAGAATTCGCGGCCGCTTCTAGATGAAGAAAGCGTTACTATTTTC
LuxP_sufffix_R	GGAGAGCTGCAGCGGCCGCTACTAGTAATTATCTGAATATCTAAATGCG
LuxPc	ATTACGCGGCCGCAGGAAACAGACCATGAAGAAAGCGTTACTATTTTCCC
LuxPd	GTAATGTCGACTCAATTATCTGAATATC
LuxP-seq-FW	CCCGTCCTGCCAGTGAGC
LuxQa	ATCGACCATGGGCAATAAATTTCGCTTAGC
LuxQc	GTAATGGATCCTTAGTGGAGGCTTGAGCC
LuxQTar_1a	CACAAATCATTGCCAATGAACGTATGTTGCTTACTCCGCTGG
LuxQTar_1b	CCAGCGGAGTAAGCAACATACGTTCATTGGCAATGATTTGTG
LuxQTar_2a	CTTAGCGACCATGAGCCATGAGTTTGCCCAGTGGCAACTGGC
LuxQTar_2b	GCCAGTTGCCACTGGGCAAACTCATGGCTCATGGTCGCTAAG
LuxS mutBbaIR	CCACACCCACTTCTAGGATGTTCTTCGCGATTTGC
LuxS mutXbaIF	GCAAATCGCGAAGAACATCCTAGAAGTGGGTGTGG
LuxS_mut_fw	CATCCTTTCTGAGAAAGGCATTCATACATTAGAGC
LuxS_mut_rev	GCTCTAATGTATGAATGCCTTTCTCAGAAAGGATG
LuxS_prefix_F	GGAGAGGAATTCGCGGCCGCTTCTAGATGGGCAATGCACCAGCGGTTCG
luxS_suffix_R	GAGGGACTGCAGCGGCCGCTACTAGTAGTCGATGCGTAGCTCTCTCAGC
LuxSa	ATCAGTCCATGGGCAATGCACCAGCGGTTCG
LuxSb	GTAATGGATCCTTAGTCGATGCGTAGC
mut_insert_fw	CTGAGGGGACGGTACCTCTACATTTACAGCTAGCTCAG
mut_insert_rv	CTGAGCTAGCTGTAAATGTAGAGGTACCGTCCCCTCAG
mut_insert2_fw	GGCAGGCGGGGCGTAATCTATAGGATCCGGCTAATAAAGG
mut_insert2_rv	CCTTTATTAGCCGGATCCTATAGATTACGCCCCGCCTGCC
mut_kpn1_pBlue	CGAGGGGGGGCCCGGTTCCCAATTCGCCCTATAG
mut_kpn1_pBlue	CTATAGGGCGAATTGGGAACCGGGCCCCCCCTCG
oriT_pre	GAATTCGCGGCCGCTTCTAGAGGACAGGCTCATGCCGGCCGC
oriT_RP4_fw	CTCGTTTCTAGAACTAGTGACAGGCTCATGCCGGCCGC
oriT_RP4_rv	TATTCGGGTACCGTCCCCTCAGTTCAGTAATTTCCTGC
oriT_suf	CTGCAGCGGCCGCTACTAGTAGTCCCCTCAGTTCAGTAATTTCCTGC
T9002_Lux_pR_rv_SpeI_BamHI_RBS	TATATACTAGTGGATCCGGTTCTGTTTCCTCTCTAGTATTTATTCGAC
T9002_LuxpR_Not_Eco_Xba_G_fw	TACGAGAATTCGCGGCCGCTTCTAGAGTCCCTATCAGTGATAGAGATTG
Term_new_fw	TACGAAAGCTTCCAGGCATCAAATAAAACGAAAGG
Term_new_rv	TATATGAGCTCTATAAACGCAGAAAGGCCCACCC
VF2	TGCCACCTGACGTCTAAGAA
VIC121	TTTATCGCAACTCTCTACTG
VIC122	CTGATTTAATCTGTATCAGG
VIC131	ATGTGTGGAATTGTGAGCGG
VIC132	CTGATTTAATCTGTATCAGG
VR	ATTACCGCCTTTGAGTGAGC

Phages

Name	application	reference

Lambda cI mut	cI deleted	W. Reiser, ZMBH
Lambda cI857	heat inducible	MBI Fermentas

Bacteria

E.coli strain	usage	reference

DH5a	amplification of plasmids	Invitrogen
HCB33	swarm assays	V. Sourjik, ZMBH
MG1655	swarm assays	V. Sourjik, ZMBH
TOP10	amplification of plasmids	Invitrogen
UU1250	chemotaxis receptor knock out	V. Sourjik, ZMBH

Bacteria Growth Media

Luria Broth (LB)	10 ^g/_l	tryptone
	5 ^g/_l	yeast extract
	10 ^g/_l	NaCl

Luria Broth (LB) plus	10 ^g/_l	tryptone
	5 ^g/_l	yeast extract
	20 ^g/_l	NaCl

TB	10 ^g/_l	bacto tryptone
	5 ^g/_l	NaCl

Standard I	15.6 ^g/_l	peptone P
	2.8 ^g/_l	yeast extract
	5.6 ^g/_l	NaCl
	1 ^g/_l	D-glucose

Minimal medium	9.8 %	M63 salts
	0.2 %	glycerol
	0.1 ^g/_l	Thiamine
	1 mM	MgSO₄
	0.8 %	amino acid mix

M9	20 %	M9 salts
	2 mM	MgSO₄
	0.4 %	glucose
	0.1 mM	CaCl₂

For cultivation of phages the media were supplied with 0.2 % maltose and 10 mM MgSO4. For preparation of agar plates 15 g/l agar, for preparation of top agar 7 g/l agar was added prior the autoclaving. For selection of resistant bacteria following antibiotics were added during cooling of at agar at about 50 °C:

Antibiotic	concentration

Ampicillin	100 µg/ml
Chloramphenicol	35 µg/ml
Kanamycin	50 µg/ml

The same concentrations of antibiotics were used for selection of resistant in media.

Methods

Preparing chemically competent cells

First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl₂. After addition of further 190 ml 100 mM CaCl₂ the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage.

Transformation of bacteria

For enrichment of vectors, E .coli TOP10 and DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 200 ng DNA solution added (depending on the concentration of the DNA solution). After a 20 minute incubation on ice, cells were made permeable for the DNA by heat shocking for 45 seconds at 42 °C and a further 2 minute incubation on ice. The samples were than rescued by adding 250µl preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shacking for induction of the antibiotic resistance. The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.

Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)

For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions. For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg. For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop.

DNA amplification using polymerase chain reaction (PCR)

By using PCR smallest amount of DNA can be detected and amplified. The principle of PCR is the selective amplification of any region of the DNA. Nevertheless, the sequence at both ends must be known for the binding of two complementary primers. The DNA region of interest is than amplified exponentially while the reproduction of the DNA takes place in three temperature stpes: denaturation of parental DNA at high temperature (95-98 °C), hybridisation of primers (58-65 °C) and DNA elongation (72 °C). For the amplification a heat-stable DNA polymerase I with a 3’-5’ exonuclease activity (proof reading) is used such as Pfu (from Pyrococcus furiosus) and Phusion (a Pyrocuccus-like enzyme) or the non proof reading enzyme Taq (from Thermus aquaticus). Phsuion polymerase was used in a 2x master mix adding only primers (20pmol each) and DNA template (~10ng) or alternatively colonies from agar plates (1-5 colonies) in a final volume of 50 µl. For taq and Pfu polymerase following reaction batch was commonly used:


1 µl template (10 ng) / colonies
2 µl primer 1 (10 pmol/µl)
2 µl primer 2 (10 pmol/µl)
1 µl polymerase (2,5 Units)
1 µl dNTP mix (10 mM)
5 µl 10x buffer
38 µl dH2O

The PCR procedure was as follows:


Initiale denaturation	95- 98 °C, 3-5 min	1 cycle

denaturation	95-98 °C, 15 sec - 1 min
annealing	58-65 °C, 15 sec - 1 min	25-28 cycles
elongation	72 °C, 15 sec - 3 min

termination	72 °C, 5 – 10 min	1 cycle
	4 °C	forever

For site directed mutagenesis PCR Pfu turbo polymerase (Stratagene) was used in the same reaction batch described above. The temperature program was as follows:


initiale denaturation	95 °C, 30 sec	1 cycle

denaturation	95 °C, 30 sec
annealing	55 °C, 1 min	16 cycles
elongation	68 °C, 2 min per kb of plasmid

termination	68 °C, 10 min	1 cycle
	4 °C	forever

For point mutagenesis primers with around 33 bases were used having the base to be altered in the middle. The melting temperature of these primers was always over 78 °C. If this melting temperature could not be achieved using 15 unchanged nucleotides at both sites the flanking arms were enlarged properly. After the PCR reaction 10 units of DpnI was added directly to the PCR tube, mixed and incubated for 1-5 h at 37 °C. DpnI digests the parental methylated plasmid DNA leaving only the mutated one. After purification using QIAquick PCR Purification Kit the plasmid was transformed as described above.

Purification of DNA from PCR reactions

PCR products were purified by the QIAquick PCR Purification Kit from Qiagen following the instructions of the Qiagen Handbook. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop.

Enzymatic hydrolysis of DNA by restriction enzymes

The restriction digest of DNA was used for analysis of purified DNA form mini or maxiprep or for isolation of specific DNA fragments for further cloning. Analytical digestions were routinely conducted in 20 µl volume. In all digestions a minimum of 2 Units restriction enzyme(s) was used per microgram DNA. Optimised buffer conditions were secured by using NEB buffer system. The final reaction volume was achieved by adding H₂O dest. and the sample was incubated at optimal temperature for the restriction enzyme(s) (normally 37 °C). Preparative digestions were conducted in a volume of 50 µl. For analysis and preparation DNA loading dye was added to the samples and they were loaded on a agarose gel or alternatively purified by QIAquick PCR Purification Kit from Qiagen.

Agarose gel electrophoresis for separating DNA

In the agarose gel electrophoresis a mixture of DNA fragments with different sizes are separated in an electrical field by their size. This is achieved by moving the negatively charged DNA through an agarose matrix while shorter fragments will run faster. The size of the pores can be controlled by agarose concentration. The higher the agarose concentration the smaller the pores are and the smaller fragments can be separated. Agarose concentrations between 0.7 and 1.5 % agarose in 0.5x TE buffer were used. The agarose was dissolved completely by heating up and 0.1 µg/ml ethidium bromide was added. The DNA fragments were separated using a constant voltage between 80 and 130 V. Under UV light (λ = 254 nm) DNA is visible through the unspecific intercalated ethidium bromide and can be documented or cut out and extracted from the gel.

Isolation of DNA fragments from an agarose gel

Plasmid DNA and DNA fragments were extracted using the Gel Extraction Kit from Qiagen following the manufacture instructions. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop.

Ligation of dsDNA fragments

T4 DNA ligase was used to form covalent phosphodiesterbonds between doublestranded DNA fragments having blunt or compatible sticky ends. For the ligation the restricted vector -DNA and the insert were mixed 1:3 or 1:5 in a total ligationvolume of 20 µl. This mixture was incubated in ligation buffer using 5 Weiss units T4 DNA ligase over night at 16 °C or at room temperature for 20 minutes and afterwards used to transform chemical competent cells.

Lysing of bacteria by ultrasonication

For the lysis of bacteria an ultrasonic tip was used. The 10-15 ml of bacteria over night cultures were sonicated with an ultrasonic tip three time for 15 seconds. After sonication cell extract was observed under microscope.

Glycerol stock

To store bacteria for long term glycerol stocks were used. Therefore 1 ml of an over night culture were added to 150 µl of 80 % Glycerol into a cryo tube, vortexed and incubated at room temperature for 30 min. Afterwards the glycerole stock was stored at -80 °C.

Preparation of plating bacteria for bacteriophage experiments

A overnight culture of the appropriate E. coli strain was grown in LB medium containing 10 mM MgSO₄ and 0.2 % maltose at 30 °C to reduce the amount of cell debris in the medium. The added maltose leads to a substantial induction of the maltose operon including the lamb gene, which encodes the cell surface receptor to which bacteriophage λ binds. After harvesting the cells they were resuspended in 10 mM MgSO₄ and diluted to a final concentration of 2.0 OD₆₀₀. The suspension of plating bacteria was stored at 4 °C for up to 1 week.

@@ Line 1,078: / Line 1,078: @@
 |-
 |}
+For point mutagenesis primers with around 33 bases were used having the base to be altered in the middle. The melting temperature of these primers was always over 78 °C. If this melting temperature could not be achieved using 15 unchanged nucleotides at both sites the flanking arms were enlarged properly. After the PCR reaction 10 units of DpnI was added directly to the PCR tube, mixed and incubated for 1-5 h at 37 °C. DpnI digests the parental methylated plasmid DNA leaving only the mutated one. After purification using QIAquick PCR Purification Kit the plasmid was transformed as described above.
+===Purification of DNA from PCR reactions===
+PCR products were purified by the QIAquick PCR Purification Kit from Qiagen following the instructions of the Qiagen Handbook. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop.
+===Enzymatic hydrolysis of DNA by restriction enzymes===
+The restriction digest of DNA was used for analysis of purified DNA form mini or maxiprep or for isolation of specific DNA fragments for further cloning. Analytical digestions were routinely conducted in 20 µl volume. In all digestions a minimum of 2 Units restriction enzyme(s) was used per microgram DNA. Optimised buffer conditions were secured by using NEB buffer system. The final reaction volume was achieved by adding H<sub>2</sub>O dest. and the sample was incubated at optimal temperature for the restriction enzyme(s) (normally 37 °C). Preparative digestions were conducted in a volume of 50 µl. For analysis and preparation DNA loading dye was added to the samples and they were loaded on a agarose gel or alternatively purified by QIAquick PCR Purification Kit from Qiagen.
+===Agarose gel electrophoresis for separating DNA===
+In the agarose gel electrophoresis a mixture of DNA fragments with different sizes are separated in an electrical field by their size. This is achieved by moving the negatively charged DNA through an agarose matrix while shorter fragments will run faster. The size of the pores can be controlled by agarose concentration. The higher the agarose concentration the smaller the pores are and the smaller fragments can be separated. Agarose concentrations between 0.7 and 1.5 % agarose in 0.5x TE buffer were used.
+The agarose was dissolved completely by heating up and 0.1 µg/ml ethidium bromide was added.
+The DNA fragments were separated using a constant voltage between 80 and 130 V. Under UV light (λ = 254 nm) DNA is visible through the unspecific intercalated ethidium bromide and can be documented or cut out and extracted from the gel.
+===Isolation of DNA fragments from an agarose gel===
+Plasmid DNA and DNA fragments were extracted using the Gel Extraction Kit from Qiagen following the manufacture instructions. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop.
+===Ligation of dsDNA fragments===
+T4 DNA ligase was used to form covalent phosphodiesterbonds between doublestranded DNA fragments having blunt or compatible sticky ends. For the ligation the restricted vector -DNA and the insert were mixed 1:3 or 1:5 in a total ligationvolume of 20 µl. This mixture was incubated in ligation buffer using 5 Weiss units T4 DNA ligase over night at 16 °C or at room temperature for 20 minutes and afterwards used to transform chemical competent cells.
+===Lysing of bacteria by ultrasonication===
+For the lysis of bacteria an ultrasonic tip was used. The 10-15 ml of bacteria over night cultures were sonicated with an ultrasonic tip three time for 15 seconds. After sonication cell extract was observed under microscope.
+===Glycerol stock===
+To store bacteria for long term glycerol stocks were used. Therefore 1 ml of an over night culture were added to 150 µl of 80 % Glycerol into a cryo tube, vortexed and incubated at room temperature for 30 min. Afterwards the glycerole stock was stored at -80 °C.
+===Preparation of plating bacteria for bacteriophage experiments===
+A overnight culture of the appropriate ''E. coli'' strain was grown in LB medium containing 10 mM MgSO<sub>4</sub> and 0.2 % maltose at 30 °C to reduce the amount of cell debris in the medium. The added maltose leads to a substantial induction of the maltose operon including the lamb gene, which encodes the cell surface receptor to which bacteriophage λ binds. After harvesting the cells they were resuspended in 10 mM MgSO<sub>4</sub> and diluted to a final concentration of 2.0 OD<sub>600</sub>. The suspension of plating bacteria was stored at 4 °C for up to 1 week.