For most cloning approaches standard methods were used as described (Sambrook et al., 1989).
3.9.1 Isolation of chromosomal DNA from yeast
Genomic chromosomal DNA (gDNA) from yeast cells was prepared using phenol/chloroform extraction (Rose et al., 1990).
Detergent Lysis Buffer pH 8.0 100 mM NaCl 10 mM Tris/HCl 1 mM EDTA
2 % (v/v) Triton X-100 1 % (w/v) SDS
TE-Buffer pH 8.0 10 mM Tris/HCl
1 mM EDTA
A saturated 10 ml liquid culture was harvested, washed once with water and the cell pellet was disrupted with 200 µl detergent lysis buffer, 200 µl phenol/chloroform/isoamylalcohol (25:24:1) (P/C/I) and 200 µl glass beads by using the FastPrep-24 machine at 4.5 m/s, 20 s for 3 times. After addition of 200 µl TE-buffer and intense mixing the aqueous phase was separated by centrifugation (21,000 g, 5 min). The upper, aqueous phase was washed twice with 200 µl P/C/I and once with C/I before the DNA was precipitated by addition of 1 ml 100 % ethanol and 6 µl 7.5 M ammonium acetate. To pellet the DNA, the solution was centrifuged 30 min at 21,000 g, 4 °C. Afterwards, the pellet was washed with 70 % ethanol, air dried and resuspended in 100 µl deionized water. DNA was stored at -20 °C for further use.
3.9.2 Isolation of plasmid DNA from E. coli
Plasmid DNA from E. coli was extracted using the commercially available Macherey-Nagel kits. Small amounts from 5 ml cultures were purified using the NucleoSpin Plasmid kit.
Isolation of larger amounts from 100-200 ml cultures was performed using the NucleoBond PC 100 kit. Plasmids were eluted in deionized water, brought to a concentration of 1 µg/µl and stored at -20 °C.
3.9.3 Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) was used to amplify DNA fragments with gene specific oligonucleotides (primers) from plasmids or gDNA. Analytical PCRs were run to verify a certain genotype or cloning step and performed using the DreamTaq polymerase. For products
Materials and Methods
45 that were subsequently used for cloning approaches proofreading polymerases like Velocity (Bioline) or Q5 (NEB) were chosen. Each of the polymerases, templates and primers required slightly different reaction conditions and in general manufacturer’s instructions were followed.
Still each PCR has in principle the same setup, which is shown for a DreamTaq PCR below.
25 µl DreamTaq PCR reaction PCR program
1 x DreamTaq buffer 95 °C 3 min Initial denaturation
0.2 mM dNTPs 95 °C 30 s Denaturation
1 µM Forward primer x °C 30 s Annealing x cycles 1 µM Reverse primer 72 °C 1 min/kb Extension
50 ng Template DNA 72 °C 10 min Final extension 1.25 U DreamTaq polymerase
Annealing temperature depended on the melting temperature of the primers. The length of the extension time was selected according to the product size.
3.9.4 Digestion with restriction enzymes
Analytical restriction digest of plasmids was performed to verify cloning steps. Plasmids or PCR products were digested to produce compatible ends to use them in cloning approaches.
Both were realized following manufacturer’s instructions and in general prepared as described below.
20 µl Restriction digest reaction 1x Enzyme specific buffer 1 µl Restriction enzyme 1-5 µg DNA
3.9.5 Agarose gel electrophoresis and DNA extraction
Agarose gels were used to analyze analytical PCRs or to purify DNA fragments from PCRs or restriction digests.
TEA-Buffer pH 8.5 40 mM Tris acetate 1 mM EDTA 6x DNA loading dye 10 mM Tris pH 7.5
60 % (v/v) Glycerol
0.03 % (w/v) Bromophenol blue 0.03 % (w/v) Xylene cyanol
46 For standard applications gels with 1 % (w/v) agarose in 1x TAE buffer were used. Agarose solution was microwaved until completely dissolved and the chilled solution was supplemented with 0.5 µg/ml ethidium bromide to stain the DNA. Gels were cast in trays with combs fitting in size to the application (analysis or purification). Samples were mixed with 6x loading dye, loaded onto the gel and let run in 1x TAE at 120 V for 30-60 min. A DNA marker served as a size control. The separated DNA fragments were visualized using UV-light and pictures taken with the INTAS system. Fragments that were run to purify were cut out of the gel with a scalpel and extracted with the peqGOLD Gel extraction kit (Peqlab) according to manufacturer’s instructions. Concentration of the eluted DNA was measured with the NanoDrop spectrophotometer.
3.9.6 Ligation of DNA fragments
Ligation of compatible ends of plasmid fragments and/or PCR products was used to create new plasmids. The purified DNA fragments were mixed in relation to their size. In general, 100 ng vector backbone was combined with 2-fold excess of insert, 1x Ligase buffer and 1 µl T4 DNA Ligase in a total volume of 10 µl. The mixture was incubated at 16 °C overnight. During this time, the ligase connects the 5’- phosphate of the vector with the 3’- hydroxyl group of the insert. The ligated plasmid was used to transform E. coli cells and subsequently selected for positive clones.
3.9.7 Gibson Assembly (GA)
Beside standard ligation Gibson Assembly was used to generate new plasmids. This method (described in (Gibson, 2011; Gibson et al., 2009)) allows joining of multiple DNA fragments with compatible overhangs in one reaction by utilizing three enzymatic activities.
2x GA master mix 5 % (v/v) PEG 8000
100 mM Tris/HCl pH 7.5 10 mM MgCl2
10 mM DTT 200 µM dNTPs 1 mM NAD
0.004 U/µl T5 exonuclease
0.025 U/µl Phusion DNA polymerase 4 U/µl Taq DNA ligase
Materials and Methods
47 5’- ends of double stranded fragments were digested by the T5 exonuclease leaving 3’- overhangs. These overhangs allow complementary annealing of the fragments.
Subsequently, 3’ gaps in the sequence are filled up by the Phusion DNA polymerase. The resulting free ends of each strand are ligated by the Taq DNA ligase. A 20 µl reaction contained 1x GA master mix, 100 ng of vector and 2-3-fold excess of the insert. After 1-3 h incubation at 50 °C the newly ligated plasmid was used to transform E. coli cells.
3.9.8 Sequencing
New plasmids were sequenced to verify the correct position and DNA sequence of the insert.
200-500 ng of the plasmid together with 20 pmol of a gene specific primer were mixed in 14 µl and send to LGC Genomics (Berlin/Germany) for sequencing.