sábado, 8 de março de 2008

20º Aula Prática

Esta aula realizada no dia 27 de Fevereiro, foi uma ida ao Laboratório Aberto do IPATIMUP no Porto; e como tal foi-nos pedido que realizassemos o seguinte relatório da actividade laboratorial:



1. Introdução:
Esta actividade realizada no Porto idealizada pelo grupo B da disciplina de Área de Projecto, com a finalidade de dar a conhecer à turma o papel das biotecnologias na saúde e na sociedade; esta actividade foi integrada na disciplina de biologia pois tal matéria está muito relacionada com a matéria leccionada.
Nas actividades utilizamos processos já dados na sala de aula, como a PCR, a electroforese, etc, tendo como objectivo uma melhor percepção e compreensão da matéria.

2.Procedimento experimental:
2.1. Material:
Tubo com inicial do alimento a tratar
Micropipeta
500 ul de InstaGene
Tubo screwcap
2g do alimento teste
Bloco térmico
Centrifugador
Tubos de PCR
Microtubos de PCR
Termociclador
Tina da electroforese
Placa de gelatina

2.2. Metodologia:
Esta actividade teve uma parte inicial em que nos foi mostrado uma apresentação de diapositivos onde a coordenadora Dora nos explicou todo o procedimento que realizaríamos no laboratório, onde nos explicaram o que eram o Laboratório Aberto, sendo este uma iniciativa do IPATIMUP para atender escolas; posteriormente explicaram o que eram alimentos transgénicos dando-nos alguns exemplos como o milho em que lhe inseriam genes para destruir potenciais parasitas, no tomate impedindo-o de apodrecer no mercado e a soja que fica assim resistente a parasitas. Depois foi-nos explicado todo o procedimento que decorreria no laboratório.
Inicialmente foi-nos explicado como trabalhar com algum material, que nos era desconhecido e necessitava de cuidados especiais, como o caso da micropipeta. Relativamente à experiência em si, após identificarmos o tubo com o nosso alimento em teste (no meu caso, a soja), agitamos vigorosamente o frasco com a solução InstaGeneTM antes de pipetar com a micropipeta, adicionando 500 ul de InstaGene ao respectivo tubo screwcap e colocá-lo posteriormente no suporte. Depois colocamos 10ml de agua destilada ao alimento teste, esmagando-o no almofariz, maceando por 2 minutos. Posteriormente pipetamos 50ul do preparado para o respectivo tubo scewcap, agitando-o depois. Então, incubamos os tubos screwcap no bloco térmico (95ºC – 100ºC) por cerca de um minuto, centrifugando depois os tubos por cerca de um minuto tendo o cuidado da centrifugadora ficar equilibrada.
Com o segundo passo principal procedemos à amplificação do DNA por PCR, onde nos seus tubos já se encontravam 20 ul de MMP (primmer verde) e de MMG (primmer vermelho), transferindo depois 20 ul de sobrenadante para o correspondente microtubo de PCR, tendo o cuidado de utilizar sempre pontas novas. Accionamos o êmbolo da micropipeta misturando duas a três vezes o conteúdo de cada microtudo de PCR, fechando convenientemente o microtubo. Posteriormente colocamos os microtubos de PCR no termociclador.
O terceiro passo principal desta actividade é o carregamento dos produto de PCR e a corrida da electroforese, em que carregamos os poços por uma ordem específica e previamente planejada para haver uma melhor interpretação dos resultados, depois fechamos a tina da electroforese correctamente e ligamos a fonte de alimentação, programando-a para uma corrida de 100V durante 40 minutos, desligando-a posteriormente, (no nosso caso, observamos resultados de uma actividade anterior, pois não disponibilizávamos do tempo necessário para o procedimento completo). Na realização da electroforese obtivemos a banda do gene PSII de 455 pd, e sendo as amostras vegetais viáveis podemos obter então uma banda de 200 pb onde verificamos posteriormente no resultado a existência ou não de alimentos transgénicos.

A turma foi dividida em dois grupos, enquanto um grupo foi realizar actividade laboratorial com a coordenadora Dora, o outro grupo foi dividido em duas equipas, com o coordenador Leandro, realizando um debate em que o tema era o consumo ou o não consumo dos transgénicos. Para nos ajudar ao debate foi distribuído umas folhas com algumas notícias sobre transgénicos e o debate teve a duração de 50 minutos.

2.3. Resultados:

3. Discussão dos resultados:
Com a tabela que nós visualizamos (representada acima) poderemos concluir que o alimento transgénico é a soja, pois é esta que tem uma semelhança de padrões através da electroforese se compara como controlo positivo (alimento transgénico), e comparando-se o milho o e tomate com o controlo negativo (alimento não-transgénico) vimos que estes tem a mesma disposição de “linhas” logo são alimentos não transgénicos.

4. Conclusões:
Com toda esta actividade foi-nos possível entender de uma melhor forma e mais correcta grande parte da matéria leccionada nas aulas, como o PCR e a electroforese, entre outros, pois o contacto físico e a prática deixa-nos perceber pormenores que infelizmente na teoria não nos é possível perceber.
Podemos ver que o esmagamento do alimento teve como finalidade permitir a dissolução dos restantes componentes da experiência em solução; percebemos também muito melhor a PCR e todas as suas fases que a constituem: desnaturação (94-95ºC), a separação das cadeias, a amplificação (50-65ºC) e extensão dos primmers (72ºC); a electroforese, técnica esta pela qual se obtem o movimento de partículas com a carga através da aplicação de uma diferença de potencial; foi outro aspecto bastante interessante pois conseguimos ver a carga eléctrica que actua na electroforese e perceber muito melhor todo este processo, até pela observação dos resultados onde foi possível perceber claramente o DNA fingerprints.

5. Referências bibliográficas / Fontes informativas:
Protocolo “O Rastreio dos transgénicos” cedido pelo Laboratório Aberto
Ficha formativa realizada no Laboratório Aberto
Apontamentos pessoais

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