enzima reazione -proteine

C4. Valutazione quantitativa e qualitativa dell’RNA totale (Protocollo C1)
mediante RT-PCR
Reazione di Retrotrascrizione (RT):
La reazione di retrotrascrizione con primers di sequenza casuale (Random primers) consente la
produzione di cDNA che verrà poi utilizzato nella reazione di Real Time PCR per determinare
la quantità relativa di specifici trascritti.
È fondamentale preparare due reazioni, una a cui verrà aggiunto l’enzima trascrittasi inversa
(RT) e un controllo negativo senza enzima. Eventuale presenza di prodotti di PCR in questo
secondo campione indicherebbe una contaminazione della preparazione di RNA da parte di
DNA genomico.
Preparare due provette Eppendorf (RT+/RT)
X entrambe le provette:
Campioni
RNA (ca. 1g come determinati da
lettura spettrofot.)
1l random primers (100ng/l)
1l dNTP’s mix (10mM)
H2O MilliQ
Totale









l
x
1
1
x
20 l
Incubare 5 ’ 65°C
Mettere i campioni in ghiaccio e centrifugare per pochi secondi
Aggiungere
4l di tampone “first strand buffer” 5X

2l DTT (0,1M)
Mescolare la miscela di reazione pipettando 1-2 volte
Incubare 2’ a 42°C
Aggiungere 1l Superscript II RT (200U) (solo per gli RT+)
Incubare 10’ a 22°C
Incubare 50’ a 42°C
Incubare 15’ a 70°C
PCR di controllo cDNA:
Preparare 2 campioni
1. Campioni
cDNA (prodotto retrotrascrizione, sia
RT+/RT)
10X PCR buffer
MgCl2 (25 mM)
dNTP’s mix (10mM)
primer 16Sfw (40 pmol/l)
primer 16Srev (40 pmol/l)
Taq (Invitrogen)
H2O MilliQ
Totale:
l
1
5
3
1
1
1
0,25
50
Protocollo PCR:
Denaturazione
5 minuti
95°C
Ciclo (ripetuto 30 volte):
Denaturazione
1 minuto
95°C
Annealing
1 minuto
60°C
Elongazione
1 minuto
72°C
Elongazione finale
5 minuti
72°C
I prodotti di PCR saranno frazionati mediante EFGAg 2 % in TAE pH 8.3 e colorazione con
Bromuro di etidio* 0.5 μg/ml.
1. Campioni
Campione di prodotto PCR #____
Loading Dye 6x
Caricare tutto in gel
l
10
2
Geni target
16s rRNA (rrs)
1542bp
AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGA
AGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGA
AACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATG
GGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGA
ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGC
CGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATT
GACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAA
TTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGA
TACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACC
GGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
TAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACC
GCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGT
GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCC
TTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTC
ATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGA
CCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCA
GAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGT
AGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAAC
CTGCGGTTGGATCACCTCCTTA
Prodotto: 131 nt (72 GC= 55%)
Tm 86.8°C
Primers utilizzati:
16Sfw: tgtcgtcagctcgtgtcgtga
16SRev: atccccaccttcctccggt
fliC
(1497bp; 498 aa)
ATGGCACAAGTCATTAATACCAACAGCCTCTCGCTGATCACTCAAAATAATATCAACAAGAACCAGTCTGCGCTGTCGAG
TTCTATCGAGCGTCTGTCTTCTGGCTTGCGTATTAACAGCGCGAAGGATGACGCAGCGGGTCAGGCGATTGCTAACCGTT
TCACCTCTAACATTAAAGGCCTGACTCAGGCGGCCCGTAACGCCAACGACGGTATCTCCGTTGCGCAGACCACCGAAGGC
GCGCTGTCCGAAATCAACAACAACTTACAGCGTGTGCGTGAACTGACGGTACAGGCCACTACCGGTACTAACTCTGAGTC
TGATCTGTCTTCTATCCAGGACGAAATTAAATCCCGTCTGGATGAAATTGACCGCGTATCTGGTCAGACCCAGTTCAACG
GCGTGAACGTGCTGGCAAAAAATGGCTCCATGAAAATCCAGGTTGGCGCAAATGATAACCAGACTATCACTATCGATCTG
AAGCAGATTGATGCTAAAACTCTTGGCCTTGATGGTTTTAGCGTTAAAAATAACGATACAGTTACCACTAGTGCTCCAGT
AACTGCTTTTGGTGCTACCACCACAAACAATATTAAACTTACTGGAATTACCCTTTCTACGGAAGCAGCCACTGATACTG
GCGGAACTAACCCAGCTTCAATTGAGGGTGTTTATACTGATAATGGTAATGATTACTATGCGAAAATCACCGGTGGTGAT
AACGATGGGAAGTATTACGCAGTAACAGTTGCTAATGATGGTACAGTGACAATGGCGACTGGAGCAACGGCAAATGCAAC
TGTAACTGATGCAAATACTACTAAAGCTACAACTATCACTTCAGGCGGTACACCTGTTCAGATTGATAATACTGCAGGTT
CCGCAACTGCCAACCTTGGTGCTGTTAGCTTAGTAAAACTGCAGGATTCCAAGGGTAATGATACCGATACATATGCGCTT
AAAGATACAAATGGCAATCTTTACGCTGCGGATGTGAATGAAACTACTGGTGCTGTTTCTGTTAAAACTATTACCTATAC
TGACTCTTCCGGTGCCGCCAGTTCTCCAACCGCGGTCAAACTGGGCGGAGATGATGGCAAAACAGAAGTGGTCGATATTG
ATGGTAAAACATACGATTCTGCCGATTTAAATGGCGGTAATCTGCAAACAGGTTTGACTGCTGGTGGTGAGGCTCTGACT
GCTGTTGCAAATGGTAAAACCACGGATCCGCTGAAAGCGCTGGACGATGCTATCGCATCTGTAGACAAATTCCGTTCTTC
CCTCGGTGCGGTGCAAAACCGTCTGGATTCCGCGGTTACCAACCTGAACAACACCACTACCAACCTGTCTGAAGCGCAGT
CCCGTATTCAGGACGCCGACTATGCGACCGAAGTGTCCAATATGTCGAAAGCGCAGATCATCCAGCAGGCCGGTAACTCC
GTGTTGGCAAAAGCTAACCAGGTACCGCAGCAGGTTCTGTCTCTGCTGCAGGGTTAA
prodotto: 61 nt (35 GC= 57.4%)
Tm 82.8°C
fw: TGCCGCCAGTTCTCCAA
rev: GACCACTTCTGTTTTGCC
csgB
456 nt (151 aa)
ATGAAAAACAAATTGTTATTTATGATGTTAACAATACTGGGTGCGCCTGGGATTGCAGCCGCAGCAGGTTATGATTTAGC
TAATTCAGAATATAACTTCGCGGTAAATGAATTGAGTAAGTCTTCATTTAATCAGGCAGCCATAATTGGTCAAGCTGGGA
CTAATAATAGTGCTCAGTTACGGCAGGGAGGCTCAAAACTTTTGGCGGTTGTTGCGCAAGAAGGTAGTAGCAACCGGGCA
AAGATTGACCAGACAGGAGATTATAACCTTGCATATATTGATCAGGCGGGCAGTGCCAACGATGCCAGTATTTCGCAAGG
TGCTTATGGTAATACTGCGATGATTATCCAGAAAGGTTCTGGTAATAAAGCAAATATTACACAGTATGGTACTCAAAAAA
CGGCAATTGTAGTGCAGAGACAGTCGCAAATGGCTATTCGCGTGACACAACGTTAA
prodotto: 75 nt (35 GC= 46.7%)
FW: CATAATTGGTCAAGCTGGGACTAA
REV: GCAACAACCGCCAAAAGTTT
http://genolist.pasteur.fr/Colibri/
Opzione: SEARCH PATTERN (allow 1 mismatch)
Tm 79.7°C