Team:Saarland/Test
From 2014.igem.org
Methods
1. Cloning
1.1 Restriction and phosphatase treatment of plasmid DNA
Originally found in bacteria, endonucleases belong to a class of enzymes that have the ability to cleave the phosphodiester bond of DNA molecules by hydrolysis. Depending on the type they recognize site specific base sequences of 4, 6 or 8 bases that 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, in particular plasmid DNA with phosphatase is beneficial if the restriction was performed with only one restriction enzyme or if the restriction with two different restriction enzymes was unbalanced and single cut plasmid DNA is left. 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 restriction batch by default:
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 batch was incubated for 10 min at 37 °C. The success of the restriction was checked in a following 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 restriction batch by default
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 batch 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 that is to be amplified. The amplification process itself is performed by a polymerizing enzyme also referred 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 amplification of genomic DNA of Bacillus megaterium by default:
2x Q5 Master Mix | 10 µl |
10 µM Forward Primer | 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 overhangs 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 PCR reactions by default:
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 PCR reaction by default:
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. 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 PCR reaction by default:
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 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 PCR reactions by default:
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 |