Protocolli di laboratorio
Analisi D-HPLC del gene CFTR Protocollo segnalato da M. C. Rosatelli (Università di Cagliari).

Procedura:

PCR:

Volume finale = 100 ml

MgCl₂ = 2mM

dNTPs = 200mM

primers = 50pM

DNA = 200ng

EXON	PRIMERs (5’-3’)	ANNEALING TEMP.	SIZE PCR product
1	F: GCACCCAGAGTAGTAGGTCT R: TGCAAGTAGATGTGGCTCTC	55°C(t)	359 bps
2	F: GTGAATATCTGTTCCTCCTCTC R: TGGTATCAAACTCCTGGTCTC	55°C(t)	338 bps
3	F: CAGATATCTGGCTGAGTGTTTGG R: GTGTTTGGAGTTGGATTCATCC	57°C(t)	407 bps
4	F: GTGTTGTGTTGAAATTCTCAGGGT R: TCCCTTACTTGTACCAGCTCACT	57°C(t)	388 bps
5	F: TGAACCTGAGAAGATAGTAAGCTAG R: GTGCTCTGCTATACAATTGACCTT	55°C(t)	416 bps
6a	F: TGTTAGTTTCTAGGGGTGGA R: ATGCATAGACAGTCCTGGT	52°C(t)	313 bps
6b	F: CTGGCACATAGGAGGCATTTACCA R: TGCAGCATAAAATAACACCCTGGAC	57°C(t)	525 bps
7	F: AATATAGGCAGAAAGACTCT R: AGAACTGATCTATTGACTGA	52°C(t)	420 bps
8	F: TGATGAATCCTAGTGCTTGG R: TGGGATTACAACCATGAGCA	55°C(t)	471 bps
9	F: TATTTTTGATGTGTGTGTGTGTGTG R: ATTTCTCCAAAAATACCTTCCAGCACT	60°C (35 cycl)	324 bps
10	F: TCCTGAGCGTGATTTGATAA R: ATTTGGGTAGTGTGAAGGG	55°C(t)	336 bps
11	F: TTCAACTGTGGTTAAGCAATAGTG F: CATGAATGACATTTACAGCAAATGC	57°C(t)	266 bps
12	F: ATGTATAAAAGTGCTACTTCTGCA R:AAGAGGTAAAATGCAATCTATGATGG	57°C(t)	342 bps
13-5’	F: TTCACAAGGTACCAATTTAATTACT R: CTCCAGTCTGTTTAAAAGATTGTTT	57°C(t)	481 bps
13-C	F: GACTTTAGCTCAAAACTCATGGGAT R: GCCAGTGACACTTTTCGTGTG	55°C(t)	490 bps
13-3’	F: ATGACACACTCAGTTAACCAAGGT R: GCATTCTACTCAATTGCATTCTGTG	55°C(t)	230 bps
14a	F:AAATAAAACCACAATGGTGGCAT R:GTGGTTCTACTTGTTGATTTTTCAG	55°C(t)	447 bps
14b	F:TCCATTAACTTAATGTGGTCTCA R:AAACACAATCTACACAATAGGAC	52°C	284 bps
15	F: TCAGTAAGTAACTTTGGCTGC R: CCTATTGATGGTGGATCAGC	57°C(t)	390 bps
16	F: TGCTAATTCTTATTTGGGTTCTG R: ATTAATGCAATAGACAGGACTTC	55°C(t)	324 bps
17a	F: TCACTGACACACTTTGTCCA R: GAATGTCCTGTACACCAACTG	57°C(t)	489 bps
17b	F: GTTATTTGCAATGTTTTCTATGGAA R: GGAAATTTCAAAGAAATCACTTGTT	52°C(t)	422 bps
18	F: GATATGTGCCCTAGGAGAGTG R: ACAGATACACAGTGACCCTCA	55°C(t)	338 bps
19	F: AGCCCGACAAATAACCAAGTGA R: ATTGCTTCAGGCTACTGGGAT	55°C(t)	447 bps
20	F: GGCATGGTACCTATATGTCACA R: CCTATGAGAAAACTGCACTGGA	55°C(t)	357 bps
21	F: AAGAATGATACAAAGCAGACATG R: CAAAATCATTTCAGTTAGGGGTA	52°C(t)	457 bps
22	F: CAGGTAGTGGGGGTAGAGGGAT R: GTGCTATTAAGTAACAGAACATCTG	57°C(t)	295 bps
23	F: GCTGATTGTGCGTAACGCTA R: TGCAGGAACTATCACATGTGAG	55°C(t)	343 bps
24	F: GAACCTCCTTAGAGCAAAAGGACAC R: TTTCTGAGGCAGAGGTAACTGTTCC	57°C(t)	480 bps

(t) = touchdown file

DHPLC:

Esone n.	% B buffer (start-end)	Range Melting Temp
1	57-65	56®65
2	56-64	55®60
3	58-66	55®58
4	55-65	55®60
5	58-66	53®58
6a	56-64	54®62
6b	59-67	55®57
7	56-66	54®61
8	59-67	52®57
9	55-64	56®63
10	56-64	55®59
11	52-62	56®61
12	54-64	56®59
13-5’	59-67	53®59
13-C	57-67	58®64
13-3’	56-61	58®62
14a	58-66	53®57
14b	53-63	55®60
15	55-65	56®60
16	54-64	54®57
17a	57-67	54®59
17b	56-66	54®63
18	54-64	55®59
19	58-66	55®61
20	57-65	55®60
21	58-66	53®59
22	53-63	57®63
23	56-64	54®59
24	59-67	57®64