CONFIGURACIÓN DE SEGURIDAD EN EL MINI-GRID

Isolation of total RNA from PC12 cells using the acid guanidinium thiocvanate- phenol method

PC 12 cell pellet was vortexed in 10 vol of solution D (Denaturing solution contained 4M guanidinium thiocyanate, 25 mM sodium citrate pH 7, 0.5% N-

lauroyl sarcosine, and 6 -mercaptoethanol to 0.1 M was added just before use).

These reagents were added in the following order: 1 vol of 2 M sodium acetate pH4, 10 vol of phenol and 2 vol of chloroform:isoamyl alcohol (49:1). The mixture was vortexed after each addition and stored on ice for 15 min after the last

addition. It was then spun at 14 OOOg for 20 min at 4”C. The aqueous phase was transferred to a fresh tube, and crude RNA was precipitated with 1 vol of isopropanol at -20”C for 1 hr and spun at 10 OOOg for 15 min. The supernatant was removed and the pellet was re-dissolved in 3 vol of solution D and re­ precipitated with isopropanol. The precipitate was washed with 75% ethanol and dissolved in RNAase-free ddHgO, with heating to 65®C to aid dissolution.

cDNA svnthesis

The cDNA synthesis was based on the method reported by Frohman (1990) and Harvey & Darlison (1991).

Reverse transcription. The reverse transcription (RTC) mixture was assembled on ice: 2 pi of lOX RTC buffer (500 mM Tris-HCl pH 8.15 at 41°C, 60 mM MgCli- 400 mM KCl, 10 mM DTT, each dNTP at 10 mM), 40 units of

pancreatic RNAase inhibitor, 6 pi of random hexamer (lOU/ml) (Pharmacia) or 2

pi of gene-specific primer CH4 (10 pM), 1 pi of RAV-2 Reverse Transcriptase

(Amersham), and ddH2 0 to make up a final volume of 10 pi. RNA (10 pg) was

dissolved in 10 pi ddH2 0 at 65®C for 5 min, rapidly cooled on ice and added to

the RTC mixture. The reaction was incubated at 42®C for 2 hr and at 52®C for a further 30 min. The reaction volume was increased to 40 pi with TE buffer and purified through chromaspin-30 TE column (Clontech) following manufacturer's

instruction, and then finally made up to 100 pi with ddH2 0 . To re-precipitate the

cDNA, 10 pi of 3M NaOAc and 60 pi of isopropanol were added to the mixture and the reaction was left on ice for 15 min. The cDNA was recovered by centrifugation at 12 OOOg for 5 min in a microfuge, washed with 70% ethanol and

resuspended in 14 pi ddH2 0. The cDNA was now ready for dA tailing or PCR

reaction.

dA tailing. The cDNA was dA-tailed so that it could be primed by a dT- containing oligonucleotide (MGD819) during the 5'RACE reaction. The following was assembled on ice: 4 pi of 5X tailing buffer (BRL), 1 pi of 10 mM dATP, 15 units of TdT (terminal deoxynucleotidyl transferase). The cDNA synthesised earlier was boiled for 2 min and rapidly cooled on ice. This was added to the mixture

above and the reaction was incubated at 37°C for 1 hr. At the end of the period,

the enzyme was deactivated at 65®C for 5 min. The dA-tailed cDNA was then diluted to 200 pi with TE buffer.

Rapid amplification of cDNA 5' ends (5*RACE)

5'Race was carried out in two steps. The first amplification used MGD819 containing a dT tail that could anneal to the dA-tailed cDNA and the amplification was between MGD820, which primed to part of MGD819 sequence, and a chimaerin specific primer (such as CH9). The second amplification was a standard PCR reaction, using MGD821 which primed part of MGD820 and a second gene- specific primer internal to that of the first specific primer.

F irst round amplification. The PCR reaction consists of the following:

dA tailed cDNA 15 pi

lOX Taq buffer (Amersham) 10 pi

dNTP mix (1.25 mM each) 16 pi

MGD819 (0.02 pg/pl) 1 pi

Primer 1: MGD820 (0.2 pg/pl) 1 pi

Primer 2: CH9 (10 pM) 3 pi

And ddHjO to 99.5 pi.

The concentration of the stock reagents is indicated in brackets. In some PCR reactions, 1 unit of Perfect Match™ Polymerase Enhancer (Stratagene) was also added to increase the final yield of discrete amplification products.

The mixture was denatured for 5 min at 95°C, followed by 15 min at 72®C. Taq polymerase (2.5 units) was added and the reaction mixture was overlayed with 100 pi of mineral oil. The reaction was incubated for 2 h at 42°C (annealing) and then for 40 min at 72®C (extension of cDNA).

30 cycles of PCR were performed at the following temperatures and time intervals: 94®C (40 sec), 58°C (1 min), 72®C (1.5 min), and a final extension of 15 min at 72®C. The reaction was cooled to 4®C.

PCR mixture from first round 5-20 pi

lOX Taq buffer 10 pi

dNTP mix (1.25 mM) 16 pi

Primer 1: MGD821 (0.2 pg/pl) 1 pi

Primer 2: CH17 (10 pM) 3 pi

And ddHjO to 99.5 pi.

The mixture was processed by following identical procedure as described for first round of amplification. 10 pi of PCR products was analysed on 1% agarose gel.

PCR

For PCR using internal primers, 5-10 pi of the cDNA was used and the procedure was similar to 'second amplification' as described above with slight variation in PCR condition according to the Tm values of the oligonucleotides used in the reaction.

Subcloning of PCR products with TA cloning kit (Invitrogen)

The TA Cloning™ System takes advantage of the non-template dependent activity of Taq polymerase that adds single deoxyadenosines to the 3' end of all duplex molecules. These A-overhangs are used to insert the PCR product into pCR 1000 vector with a single 3' T overhang at the insertion site. Thus, PCR product was cloned directly into the vector without prior enzymatic modifications.

The ligation reaction contained 1 pi of PCR product, 1 pi of lOX ligation

buffer, 2 pi of pCR 1000 vector (25 ng/pl), 1 pi T4 DNA ligase and ddH2 0 to a

final volume of 11 pi. The reaction was incubated at 12°C overnight. 1 pi of the

ligation mixture was used for transformation with competent E.coli cells (INVaF').

The DNA-cell mixture was incubated on ice for 30 min, heat shocked for 60 sec at 42°C and rapidly cooled on ice for 2 min. The cells were expressed for 1 hr at 37®C in 400 pi SOC medium and plated on agar plate containing 50pg/ml of Kanamycin. The positive clones were selected as described in 'Identification of colonies containing recombinant plasmids' and eventually sequenced.