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Methods

1. Cloning

1.1 Restriction and phosphatase treatment of plasmid DNA

Originally found in bacteria, endonucleases belong to a class of enzymes capable of cleaving the phosphodiester bond of DNA molecules by hydrolysis. Depending on the type they recognize site specific base sequences of 4, 6 or 8 bases respectively, which are often palindromic. Treatment of different DNA molecules with the same endonucleases allows the combination of the resulting DNA fragments in a following ligation reaction.

The treatment of DNA fragments is beneficial if the restriction was performed with only one restriction enzyme. It can also be beneficial if the restriction with two different restriction enzymes was unbalanced and single cut plasmid DNA is left. This applies in particular to the treatment of plasmid DNA with phosphatase. As the nomenclature indicates the phosphatase cleaves the phosphate group at the 5´terminus of DNA fragments and thereby minimizes the background of mono ligated plasmid DNA. Consequently the amount of transformants with insert DNA in the plasmid construct is considerably increased.

The following components were used for a basic restriction mixture:

10 x FastDigest Buffer	3 µl
Restriction Enzyme 1	10 U
Restriction Enzyme 2	10 U
FastAP	1 U
Plasmid	approx. 1 µg
ddH2O	to 30 µl

The mixture was incubated for 10 min at 37 °C. The success of the restriction was subsequently checked in an agarose gel electrophoresis.

1.2 Restriction of PCR products:

Corresponding restriction sites were added in a PCR reaction at the 5´and 3´termini of insert DNA via modified primers. After gelextraction the following components were used for a basic restriction mixure.

10 x FastDigest Buffer	3 µl
Restriction Enzyme 1	10 U
Restriction Enzyme 2	10 U
PCR Product	25 µl
ddH2O	to 30 µl

The mixture was incubated for 10 min at 37 °C followed by purification via QIAquick Gel Extraction Kit (Qiagen).

1.3 Polymerase chain reaction (PCR)

The PCR is a standard procedure in molecular biology that enables the amplification of distinct DNA sequences in vitro. Short flanking DNA oligomers define the DNA sequence which is to be amplified. The amplification process itself is performed by a polymerizing enzyme also referred to as DNA polymerase. Since its invention in 1983 numerous variations have been established for different applications.

1.3.1 Amplification of genomic DNA:

The following components were used for the basic amplification of genomic DNA of Bacillus megaterium:

2x Q5 Master Mix	10 µl
10 µM Forward	1 µl
10 µM Reverse Primer	1 µl
Genomic DNA	100 ng
ddH2O	to 20 µl

The following speed cycle program was used:

Step	Temperature [°C]	Time [s]
1. Initial Denaturation	98	30
2. Denaturation	98	15
3. Annealing	60	15
4. Extention	75	20/kb
5. Final Extention	72	120
6. storage	4	for ever

Presence, absence and size of amplification products were determined by agarose gel electrophoresis.

1.3.2 Splicing by overlapping extension (SOE)-PCR:

The SOE-PCR is a common variation of the standard PCR reaction and primarily used for the generation of fusion genes. The name refers to the special set of primers. Their single stranded endings are complementarily overlapping with the DNA sequences of the DNA fragments to be fused. Figure 1 shows the flowchart of the SOE-PCR for the generation of a hyaluronan synthase (has2) - green fluorescent protein (gfp) fusion gene.

The following components were used for the first standard PCR reactions:

2x Q5 Master Mix	10 µl
10 µM Forward Primer a or c	1 µl
10 µM Reverse Primer b or d	1 µl
Plasmid DNA	10 ng
ddH2O	to 20 µl

The following components were used for the second standard PCR reaction:

2x Q5 Master Mix	10 µl
10 µM Forward Primer a	1 µl
10 µM Reverse Primer d	1 µl
Amplification Product 1 and 2	1:1 Molar Ratio (approx. 10 ng)
ddH2O	to 20 µl

The following speed cycle program was used:

Step	Temperature [°C]	Time [s]
1. Initial Denaturation	98	30
2. Denaturation	98	15
3. Annealing	60	15
4. Extention	75	20/kb
5. Final Extention	72	120
6. storage	4	for ever

Presence, absence and size of amplification products were determined by agarose gel electrophoresis.

1.3.3 Colony PCR:

After transformation of competent E. coli cells the colony PCR is a popular high-throughput technique in molecular biology for the quick identification of the presence, absence or orientation of insert DNA in plasmid constructs, respectively. An extended heating step at the beginning of the PCR reaction results in cell lysis and release of plasmid DNA which serves as template for the following amplification with insert specific primers. Time consuming DNA isolations and restrictions of multiple colonies are superfluous.

The following components were used for the second standard PCR reaction:

10x ThermoPol Reaction Buffer	2 µl
10 mM dNTP Mix	0,4 µl
10 µM Forward Primer	1 µl
10 µM Reverse Primer	1 µl
Taq DNA Polymerase	½ U
ddH2O	to 20 µl
colony	Resuspend

The following speed cycle program was used:

Step	Temperature [°C]	Time [s]
1. Initial Denaturation	95	180
2. Denaturation	95	30
3. Annealing	60	30
4. Extention	68	60/kb
5. Final Extention	68	300
6. storage	4	for ever

Presence, absence and size of amplification products were determined by agarose gel electrophoresis.

1.3.4 Quick Change (QC)-PCR:

The QC-PCR is a common variation of the standard PCR and useful for site-directed mutagenesis of plasmid DNA. The special design of mutagenic primers allows the insertion or deletion of whole sequences, respectively, as well as the substitution of single nucleotides for applications such as restriction site mutagenesis or amino acid exchange. Figure 2 shows the experimental setup for a QC-PCR.

The following components were used for the standard PCR reactions:

2x Q5 Master Mix	25 µl
10 µM Forward QC Primer	2,5 µl
10 µM Reverse QC Primer	2,5 µl
Plasmid DNA	10 ng
ddH2O	to 50 µl

The following speed cycle program was used:

Step	Temperature [°C]	Time [s]
1. Initial Denaturation	95	30
2. Denaturation	98	15
3. Annealing	60	35
4. Extention	72	20/kb
5. Final Extention	72	300
6. storage	4	for ever

The following components were used for digestion and removal of the template plasmid DNA :

10 x FastDigest Buffer	5 µl
DpnI	1 U
QC-PCR Batch	40 µl
ddH2O	to 50 µl

Presence, absence and size of amplification products were determined by agarose gel electrophoresis after digestion with DpnI.

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