Microbiological and Molecular Biological Methods

5.2. Microbiological and Molecular Biological Methods

5.2.1. Cultivation and Conservation of E.coli Strains

Strains were streaked out from permanent cultures to form single colonies on LB agar plates supplemented with the appropriate antibiotics. Plates were incubated at 37^◦C for 12-16 h, if not stated otherwise, and stored at 4^◦C. Single colonies were used to inoculate liquid LB medium or new agar plates. Growth in liquid cultures was achieved using glass tubes in a roller drum or Erlenmeyer flasks in a shaking incubator.

Each liquid culture was inoculated with a single cell colony and grown at 37^◦C unless stated otherwise. Growth was followed spectroscopically by determining the optical cell density at 600 nm (OD₆₀₀). Permanent cell cultures were prepared from liquid cultures grown to exponential phase by mixing 800µl cell culture with 200µl DMSO.

The mixture was immediately frozen using liquid nitrogen and subsequently stored at -80^◦C.

5.2.2. Cultivation and Conservation of Yeast Strains

S.cerevisiae strains were streaked out from permanent cultures to form single colonies on YPD plates or the appropriate selection plate when containing a plasmid. Plates were incubated at 30^◦C for 2-3 days, if not stated otherwise, and stored at RT. Single colonies were used to inoculate liquid medium or new agar plates. Growth in liquid cultures was achieved using glass tubes in a roller drum or Erlenmeyer flasks in a shaking incubator. Each liquid culture was inoculated with a single cell colony and grown at 30^◦C unless stated otherwise. Growth was followed spectroscopically by determining the optical cell density at 600 nm (OD₆₀₀). Permanent cell cultures were prepared from liquid cultures grown to exponential phase by mixing 800µl cell culture with 200µl DMSO. The mixture was immediately frozen using liquid nitrogen and subsequently stored at -80^◦C.

5.2.3. Plasmid DNA Preparation

To purify plasmid DNA the QIAprep Spin Miniprep Kit (Qiagen) was used according to the protocol provided by the manufacturer.

5.2.4. PCR Mutagenesis

Using PCR technique various DNA-fragments can be amplified. A standard PCR reaction contained 1x reaction buffer, 1 µl of DNA template (50 - 100 ng), 100 µM of dNTP mix, 0.5µM of each primer and 0.5 µl of Phusion Polymerase in 50 µl final volume. The cycling parameters for a standard PCR are shown in Table 5.6.

Table 5.6.: Cycling Parameters for PCR Segment Temperature Time

Table 5.7.:Cycling Parameters for single site directed mutagenesis

The Single Site-Directed Mutagenesis can be used to introduce single site mutations into a DNA template using polymerase chain reaction (PCR). This technique was used to generate the various cystein mutations in Zuotin. A standard ssm-PCR reaction contained 1x reaction buffer, 1 µl of DNA template, 100 µM of dNTP mix, 0.5 µM of each primer and 0.5 µl of Phusion Polymerase in 50 µl final volume. The cycling parameters for the mutagenesis are shown in Table 5.7. The reaction products were treated with the restriction endonucleaseDpnI at 37^◦C overnight, to digest the parental DNA template. The DpnI endonuclease with the target sequence: 5’-Gm6ATC-3’ is specific for methylated and hemimethylated DNA (Nelson et al., 1992). Subsequently the multiply mutated single stranded DNA was transformed into DH5α competent cells.

5.2.6. Restriction Digest

Restriction digests were performed using the corresponding restriction endonuclease. 5 - 20µl of DNA sample (plasmid or PCR product) were incubated with 3µl 10 x reaction buffer and 0.2 - 1 U of the corresponding enzyme (w/o BSA) in a total volume of 30µl.

5.2. Microbiological and Molecular Biological Methods After incubation at the enzyme-specific temperature for 5 - 16 h the digest was used

for further work.

5.2.7. Ligation

Ligases link DNA ends by catalyzing the phosphodiester-bonding. DNA fragments with matching digested ends were incubated in no more than 10 µl total volume with 40 U T4-DNA-ligase and the recommended ligation buffer. The optimal ratio of vector to insert DNA was 1:3 for “sticky end” ligations. Different conditions for incubation were used: over night at 16^◦C, 2 h at room temperature or 30 min at 30^◦C.

5.2.8. Preparation of Chemically Competent E. coli Cells

A 50 ml culture was inoculated with 0.5 ml of an overnight culture and grown in medium (LB broth with 20 mM MgSO₄, 10mM KCl) to mid-logarithmic phase. The cells were kept on ice for 10 min, pelleted at 1,500g for 10 min at 4^◦C, resuspended gently in 150 ml cold TFB1 and incubated for 15 min on ice. The cells were sedimented again at 1,500g for 10 min at 4^◦C and resuspended in 20 ml cold TFB2. The cells were aliquoted in 250 µl portions, frozen in liquid nitrogen and stored at -80^◦C.

5.2.9. Transformation of Chemically Competent E. coli Cells

For transformation, 1-5 µl DNA were incubated with 50µl competent cells for 30 min on ice, then the cells were heat-shocked for 90 s at 42^◦C and subsequently kept on ice for 2 min. 1 ml of LB medium was added and the mixture was shaken for 45 min at 37^◦C to allow bacterial recovery and expression of the antibiotic resistance gene.

The transformed cells were plated onto LB agar plates containing the appropriate antibiotics.

5.2.10. Transformation of Yeast Cells

3 µl of 10 mg/ml boiled carrier DNA and 1 µg plasmid were mixed with 100 µl transformation mix (400 mM lithium acetate, 40 % PEG-3350, 130 mM β-Me). For transformation one large yeast colony was resuspended in the mix and vortexed. The mixture was then incubated rotating for 30 min at 37^◦C. The transformed cells were

pelleted, resuspended in H₂O and subsequently plated onto agar plates containing the selective media.

5.2.11. Spot Test

Yeast cells were grown to exponential phase and adjusted to OD₆₀₀ = 0.2. Serial dilutions were spotted on agar plates containing selective media and appropriate aux-otrophy marker.

5.2.12. DNA - Gel-Electrophoresis / Agarose Gel

Agarose gel electrophoresis is used to separate DNA strands by size and can be utilize to estimate the size of the DNA. Negatively charged DNA molecules migrate through the agarose matrix using an electric field, with shorter molecules moving faster than longer ones. Usually 1% agarose gels prepared in 1xTAE electrophoresis buffer were used in a casting tray. One-sixth volume of a 6 x concentrated loading dye is added to each sample, mixed and loaded into the wells. The samples are separated at 50 -100 V (depending on the size of the gel) until the required separation is achieved. To visualize and photograph the DNA fragments, a long wave UV light box is used.

5.2.13. DNA Sequencing

All mutations were verified by DNA sequencing performed at GATC - Biotech, Kon-stanz.