cDNA synthesis

For the construction of the cDNA the λ ZAP Express cDNA Synthesis Kit (Stratagene) was used. Poly(A)⁺ RNA corresponding to 2x10⁷ LAN-1 cells dissolved in 37.5 µl DEPC-treated water were used as starting material. The following reagents were combined and gently mixed:

5 µl 10x first strand buffer

3 µl first strand methyl nucleotide mixture 2 µl linker primer (1.4 µg/µl)

1 µl RNase Block Ribonuclease Inhibitor (40 U/µl) 37.5 µl poly(A)+ RNA in DEPC-treated water

The linker primer contained a Xho I restriction site to allow a sense orientation (EcoR I → Xho I) of the finished cDNA in the λ ZAP vector with regard to the lacZ promotor. To protect the cDNA against a later following Xho I digestion dCTP was substituted against 5-methyl dCTP in the first-strand nucleotid mixture to hemimethylate the cDNA. The primers were allowed to anneal for 10 min at room temperature before 1.5 µl of MMLV-RT (50 U/µl) was added. The reaction mixture was incubated for 1 h at 37°C and then placed on ice.

During the second strand synthesis RNase H was used to nick the RNA bound to the first-strand cDNA thereby producing a multitude of fragments which served as primers for DNA polymerase I. The original mRNA was thereby translated into second-strand cDNA. The following precooled components for second strand synthesis were subsequently added:

20 µl second-strand buffer

6 µl second strand dNTP mixture 16 µl sterile distilled water

2 µl RNase H (1.5 U/µl)

11 µl DNA polymerase I (9.0 U/µl)

The reaction mixture was incubated for 2.5 h at 16°C and the tubes placed on ice afterwards. Temperatures above 16°C were not acceptable due to the risk of formation of unclonable hairpin structures. For blunting of the uneven cDNA termini 23 µl of blunting dNTP mix and 2 µl of cloned Pfu DNA polymerase (2.5 U/µl) were added and the mixture was incubated for 30 min at 72°C. The cDNA was extracted twice with phenol-chloroform [1:1 (v/v)] and precipitated overnight at -20°C with 20 µl of 3 M sodium acetate and 400 µl of 95 % ethanol. The precipitated cDNA was collected by centrifugation at 14000 rpm at 4°C for 60 min, washed once with 70 % ethanol and airdried.

To allow for directional cloning of the cDNA in the λ ZAP vector EcoR I adaptors were ligated to the blunt termini. The pellet was therefore resuspended in 8 µl of a solution containing the EcoRI adapters and incubated at 4°C for 1h to allow the complete solution of the pellet. The following components were then added:

1 µl 10 x ligase buffer 1 µl 10 mm rATP

1 µl T4 DNA ligase (4 u/µl)

The ligation reaction was performed over 2 days at 4°C. The ligase was subsequently heat inactivated at 70°C for 30 min.

After cooling the mixture to room temperature the adapters were phosphorylated by adding the following components:

1 µl 10 x ligase buffer 2 µl 10 mM rATP 6 µl sterile water

1 µl T4 polynucleotide kinase (10 U/µl)

After phosphorylation for 30 min at 37°C, the kinase was heat inactivated for 30 min at 70°C. To produce the cohesive end the EcoR I adapter and the residual linker primer from the 3´ end of the cDNA were released by a Xho I digestion. 28 µl of Xho I buffer supplement and 3 µl of Xho I (40 U/µl) were added, the mixture was incubated for 1.5 h at 37°C and the cDNA afterwards precipitated overnight by adding 5 µl of 10 x STE buffer and 125 µl of 95 % ethanol. The cDNA was collected by centrifugation at 14000 rpm at 4°C for 60 min, the dried pellet resuspended in 14 µl of 1 x STE buffer and mixed with 3.5 µl of column loading dye.

To separate the finished cDNA from the linker primer and the EcoRI adapter a size separation was done using Sepharose CL-2B gel filtration media. A 1ml pipet was used as column and connected with a tube to a syringe which served as buffer reservoir. The gel filtration matrix was equilibrated in 1x STE buffer, filled into the column until approximately 0.5 cm under the point where the pipet and the syringe joined. The matrix was thorougly washed and the cDNA sample was then applied directly on top of the column bed. Fractions were collected in 100 µl aliquots. The different fractions were afterwards extracted twice with 100 µl phenol-chloroform [1:1 (v/v)] and precipitated overnight at -20°C by adding 200 µl of 95 % ethanol. After collecting the cDNA by centrifugation, the pellet was washed once with 70% ethanol and then resuspended in 3.5 µl of sterile water. The quantity of the cDNA was subsequently checked by an ethidium bromide plate assay (see 3.5.2.), the size by running an agarose gelelectrophoresis (see 3.5.5.)

3.5.2.3. Construction of the cDNA library in the λλ ZAP Express vector

After determining the size and quantity of the cDNA in the different fractions the best suited fractions were used for ligation into the λ ZAP Express Vector using the following components:

1.0 µl λ ZAP Express Vector 0.5 µl 10 x ligase buffer 0.5 µl 10 mM rATP (pH 7.5)

1.0 µl resuspended cDNA (approximately 100 ng cDNA) 1.5 µl sterile water

0.5 µl T4 DNA ligase (4 U/µl)

The ligation reaction was performed over 2 days at 4°C.

2 µl of each ligation reaction was then added to packaging extract (Gigapack III Gold packaging extract, Stratagene) and packaging was allowed to take place for 2 hours at room temperatue. The packaging reaction was stopped by adding 500 µl of SM-buffer and 20 µl of chloroform. The cell debris was sedimented by centrifugation and the supernatant containing the phage was checked for titer as well as efficiency of the packaging process by blue white screening (see 3.5.5.).

The packaging reactions were therefore plated in the E.coli strain XL1-Blue MRF´ by adding 1 µl of the undiluted stock and 1 µl of a 1:10 dilution of the λ phage to 200 µl of the host cells (OD600 = 0.5). The various suspensions were incubated for 15 min at 37°C before the following components were added:

3 ml NZY Top Agar (melted and prewarmed to 50°C) 15 µl 0.5 M IPTG (in dH₂0)

50 µl X-gal (250 mg/ml in DMF)

The suspensions were immediately plated onto 85 mm² NZY agar plates and cooled for 15 min to harden the NZY Top Agar. The plates were then incubated overnight at 37°C.

To achieve a large stable library with a high titer the primary library was amplified by adding 5 x 10⁴ pfu to 600 µl of XL1-Blue MRF´cells at an OD₆₀₀ = 0.5 in 10 mM MgSO₄. Plating was done as described before (see above) using 8 ml NZY Top Agar and 150 mm² plates. All in all 1 x 10⁶ plaques were amplified. After overnight growth the plates were overlied with 10 ml of SM-buffer for 24 h to allow the phages to diffuse into the SM-buffer. The bacteriophage suspension was then recovered, 5 % (v/v) chloroform was added and the suspension was incubated for 15 min at room temperature to kill any residual cells. The cell debris was afterwards pelleted by centrifugation for 10 min at 2000 rpm . After addition of 0.3%

(v/v) chloroform the supernatant was aliquoted and stored at 4°C and -80°C, respectively.

3.5.2.4. Assessment of the cDNA library

To assess the quality of the constructed cDNA library regarding the size of the inserted cDNA approximately 500 plaques were plated. 10 of these plaques were picked and the phages in vivo excised (see 3.5.2.).

The resulting phagemids were plated on LB-kanamycin plates, single colonies were then picked and mini preps prepared (see 3.5.3). The isolated phagemid DNA was resuspended in 20 µl TE buffer and subjected to a restriction digestion with the restrictions enzymes Xho I and Pst I (MßB). Therefore the following components were combined and incubated for 1 h at 37 °C .

4 µl resuspended phagemid 0.5 µl Xho I

0.5 µl Pst I

1 µl 10 x buffer red 4 µl sterile water

Analysis of the restriction digest was done by agarose gelelectrophoresis.