Molecular biology

5.2.2.1 Isolation of yeast genomic DNA

YPH499 was cultured at 30^◦C in YPD overnight. 10⁸ cells (approximately 10 OD) were used to isolate genomic DNA using the “High Pure PCR Template Kit” (Roche) according to the manufacturers instructions. Briefly the cells were collected by centrifugation for 5 min at 3000 xg. The pellet was resuspended in 200µL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Zymolyase was added to 0.25 mg/mL and the suspension was incubated for 30 min at 37^◦C. Subsequently 200µL binding buffer and 40µL 1 mg/mL PK were added, the sample was mixed and incubated for 10 min at 70^◦C. After addition of 100µL isopropanol the sample was applied to the filter tube and centrifuged for 1 min at 8000 xg. The filter tube was washed with 500µL inhibitor removal buffer and 500µL wash buffer. The DNA was eluted using 200µL prewarmed elution buffer. Genomic DNA was stored at -20^◦C.

5.2.2.2 Isolation of plasmid DNA from E. coli

Plasmid DNA was isolated using the “Wizard Plus SV Minipreps DNA Purification Sys-tem” (Promega) according to the manufacturers instructions. Briefly, 2-5 mL of anE. coli culture in LB medium were harvested by centrifugation for 3 min at 12000 xg. The pellet was resuspended in 250µL resuspension buffer, 250µL lysis buffer were added along with 10µL alkaline protease. The sample was inverted 6 times and incubated at room temper-ature for 5 min. Subsequently 350µL neutralization buffer were added and the samples were centrifuged for 10 min at 20200 xg. The supernatant was loaded on a filter tube and centrifuged for 1 min, 20200 xg. The filter was washed with 750 and 250µL washing buffer, and was spun dry afterwards. The DNA was eluted by incubation of the filter with 100µL dH₂O for 1 min, followed by centrifugation for 1 min at 20200 xg. Plasmid DNA was stored at -20^◦C

5.2.2.3 PCR

DNA segments were amplified from yeast genomic DNA or plasmid DNA by the poly-merase chain reaction using KOD polypoly-merase (Novagen). To this end, reactions contain-ing 0.4µM forward and reverse primer, 10-100 ng template DNA, 1X KOD buffer, 1.5 mM

MATERIALS AND METHODS

MgSO₄, 0.2 mM of each dNTPs and 1 U KOD Hot Start DNA polymerase were prepared.

Cycling conditions were: 2 min at 95^◦C for polymerase activation, 20 s at 95^◦C, 10 s at 48-55^◦C (depending on the primer combination used) and 10 to 25 s/kb (depending on the length of the product) for cycling (35 cycles). PCR products were analyzed by agarose gel electrophoresis (1 % low melting agarose (Roth) in 1x TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA) in horizontal electrophoresis cell (BioRad) at 110 V for 30 min. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Briefly, the DNA fragment was mixed with an equal volume of binding buffer (however resulting in at least 200µL), loaded on a filter tube, incubated 1 min at room temperature and centrifuged for 1 min at 20200 xg. The filter was washed with 700 and 500µL washing buffer, and subsequently the filter was spun dry. Elution was achieved by incubation of the filter for 1 min with 50µL dH₂O and centrifugation for 1 min at 20200 xg. Purified DNA fragments were stored at -20^◦C.

5.2.2.4 Cloning

Clonging was carried out according to standard procedures (Sambrook and Russell, 2001).

In order to insert purified PCR products into plasmids, both were first digested with the appropriate restriction enzymes (FastDigest (Fermentas/ Thermo Scientific)). To this end, 30µL reactions were prepared containing 1x FastDigest buffer, 1µL of each restriction enzyme and 800-1000 ng DNA. Restriction was carried out for 30 min at 37^◦C with slight agitation (400 rpm). Optionally, the opened plasmid was dephosphorylated using 1µL alkaline phosphatase and addition of the phosphatase buffer to a final concentration of 1x to the reaction. Fragments were purified (see 5.2.2.3) and mixed: 5µL of plasmid, 10µL of insert and 4µL DNA ligation buffer and 1µL T4 DNA ligase (Rapid DNA Ligation Kit, Thermo Scientific). After incubation for 30 min at 21^◦C, 10µL of the ligation reaction were used to transform 50µL competed XL1 Blue E. coli (see 5.2.1.2). Constructs were analyzed by analytical restriction digest and sequencing (GATC Biotech AG, Konstanz, Germany).

5.2.2.5 In vitro mutagenesis

Mutations were introduced into purified plasmids using the “QuikChange Site-Directed Mutagenesis Kit” (Agilent Technologies). Partially overlapping primers containing the

MATERIALS AND METHODS

mutations were designed according to Zheng (2004). Reactions (50µL) contained 50 ng plasmid DNA, 0.2µM forward and reverse primer, 1µL dNTP mix, 1x buffer and 2.5 U PfuTurbo DNA polymerase. After a initial denaturation for 30 s at 95^◦C, cycling condi-tions were 30 s at 95^◦C, 1 min at 48-55^◦C, 1 min/kb at 68^◦C for 20 times. Subsequently the reaction was cooled down on ice for 2 min, 10 U DpnI were added in order to digest the parental DNA for 1 h at 37^◦C. 5µL of the reaction were used to transform 50µL competent XL1 Blue E. coli (see 5.2.1.2). Success of the mutation was determined by analytical restriction digestion and/ or sequencing.

5.2.2.6 In vitro transcription

In order to generate capped mRNA’sin vitro, PCR products containing a SP6 promoter in front of the ORF were transcribed using the mMESSAGE mMACHINE SP6 kit (Life Technologies). To this end, 20µL reactions were prepared, containing: 1x NTP/ CAP, 1x reaction buffer, 1µg PCR product and 2µL enzyme mix. Transcription was performed for 90 min at 37^◦C, afterwards 2 U TURBO DNaseI were added, followed by another 15 min at 37^◦C. In order to recover the mRNA, 30µL nuclease-free water and 30µL LiCl precipitation solution (7.5 M lithium chloride and 50 mM EDTA) were added, the sample was mixed and frozen at -20^◦C for at least 30 min. Subsequently, the sample was centrifuged for 15 min at 16100 xg and 4^◦C. The RNA pellet was washed with 1 mL 70 % ethanol, centrifuged and dried. Finally the RNA was resuspended in 50µL DEPC water.

mRNA was stored at -80^◦C.

5.2.2.7 In vitro translation

Translation was performed from plasmid templates (TNT^R Quick Coupled Transcrip-tion TranslaTranscrip-tion kit (Promega)) or mRNA (Flexi^R Rabbit Reticulocyte Lysate System (Promega)). For the coupled reaction 40µL TNT^R Quick Master Mix were mixed with 1µg plasmid DNA and 50µCi³⁵S-Met. For the translation from mRNA 33µL Flexi^R Rab-bit Reticulocyte Lysate, 1µL 1 mM amino acid mix without methionine, 1.5µg mRNA and 50µCi³⁵S-Met. Additionally KCl and MgAc where added to 70-120 mM and 0-2 mM, depending on the precursor synthesized (see Table 13).

MATERIALS AND METHODS

Tab.13:Conditionsforradiolabelingofdifferentprecursors. PrecursorPlasmidPCRprimersadd.MgAc[mM]add.KCl[mM] S.c.Cox5aC21SP6,T7170 S.c.DLD(1-152)-DHFRDLD-DHFRSP6,T7170 S.c.b2(167)∆-DHFRB04SP6,T7170 S.c.b2(1-220)-DHFRB18SP6,T7170 S.c.b2(220)∆-DHFRB46SP6,T7170 N.c.F1βF04SP6,T70100 S.c.Pam16pCS29M13fwd20;M13pUCrev2120 S.c.Pam17pCS30M13fwd20;M13pUCrev2100 S.c.Pam18pCS31M13fwd20;M13pUCrev2120 S.c.Tim17pCS47M13fwd20;M13pUCrev270 S.c.Tim21pCS46M13fwd20;M13pUCrev270 S.c.Tim44pCS126SP6;CS282:XbaI0120 S.c.Tim501255M13fwd;CS168270 S.c.Ssc1pCS33M13fwd20;M13pUCrev2100 S.c.Mgr2pCS62CS188,CS189270 S.c.Tom20genomicDNACS197:SacI;CS198:BamHI270 S.c.Tom40genomicDNACS199:SacI;CS200:BamHI2120

MATERIALS AND METHODS