|
|
|
|
EXON |
LENGTH (bp) |
PRIMER F |
PRIMER R |
HOT START |
DENAT |
ANNEAL |
ELONG |
fin. ext. |
cycles |
|---|---|---|---|---|---|---|---|---|---|
|
exon 1a |
350 |
LA1F |
LMNA 1aR2 |
96° 5 ' |
96° 1' |
61° 45'' |
72° 1' |
72° 7' |
35x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 1b |
332 |
LMNA1bF2 |
LMNA1bR |
96° 5 ' |
96° 1' |
61° 45'' |
72° 1' |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 2 |
339 |
LA2FB |
LA2RF |
96° 5' |
96° 1' |
57° 30'' |
72° 1' |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 3 |
232 |
LMNA3F2 |
LMNA3R2 |
96° 5' |
96° 1' |
61° 1' |
72° 1' |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 4 |
341 |
LMNA4F |
LA4R |
96° 5' |
96° 1' |
62°45'' |
72° 1' |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 5 |
231 |
LMNA5F |
LMNA5R |
96° 5' |
96° 1' |
61° 1' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 6 |
376 |
LA6F |
LA6R |
96° 5' |
96° 1' |
61° 1' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 7 |
359 |
LA7F |
LA7R |
96° 5' |
96° 1' |
63° 45'' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 8 |
205 |
LA8F |
LA8R |
96° 5' |
96° 1' |
61° 45'' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 9 |
239 |
LA9F |
LA9R |
96° 5' |
96° 1' |
61° 1' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 10 |
319 |
LMNA10F |
LMNA10R |
96° 5' |
96° 1' |
59° 1' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 10B |
214 |
LA10FB |
LA10RB |
96 |
96° 1' |
62° 45'' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 11 |
359 |
LMNA11F |
LMNA11Rev |
96° 5' |
96° 1' |
59° 1' |
72° 1 |
72° 7' |
40x |
|
|
|
|
|
|
|
|
|
|
|
|
exon 12 |
198 |
LMNA12F |
LMNA12R |
96° 5' |
96° 1' |
61° 45' |
72° 1 |
72° 7' |
40x |
|
Exon |
|
Forward primer |
Reverse primer |
|---|---|---|---|
|
|
|
|
|
|
1A |
LA1F/LMNA 1aR2 |
CCC AgA TCC CgA ggT CCg AC |
AgC CCT gCg TTC TCC gTT TCC |
|
1B |
LMNA1bF2/LMNA1bR |
ACC CgC ATC ACC Cgg CTg |
CCT CTC CAC TCC CCg CCA |
|
|
|
|
|
|
2 |
LA2FB/LA2RB |
ATT gCA ggC ATA gCA gCg C |
gAg ggC CTA ggT AgA AgA G |
|
|
|
|
|
|
3 |
LMNA3F2/LMNA3R2 |
TTC TTg TgT TCT gTg ACC CCT T |
CCC AAg TCT gTC ATC ACC CA |
|
|
|
|
|
|
4 |
LMNA4F/LA4R |
gTC CCT ggg TCT Tgg CCT CC |
gCC ACC ATC TgC CTg ATC C |
|
|
|
|
|
|
5 |
LMNA5F/LMNA5R |
CCT CCA CCC CTC CCA gTC AC |
TgC ATC Cgg CCC AgA CTC Tag |
|
|
|
|
|
|
6 |
LA6F/LA6R |
Cag CTg TCT CCT ACA CCg ACC |
ggT CTA gTC AAg gCC AgT TgC |
|
|
|
|
|
|
7 |
LA7F/LA7R |
gAg gTg CTg gCA gTg TCC TC |
CTT CTg TCT TgC CAC TCT CTC C |
|
|
|
|
|
|
8 |
LA8F/LA8R |
ACC CAA gAg CCT ggg TgA gC |
CAC CCA Agg TCT CCC CAg Ag |
|
|
|
|
|
|
9 |
LA9F/LA9R |
TTg ggT gTC gAT ggg AgC gC |
gCA gCT gCC TCC gAT gTT gg |
|
|
|
|
|
|
10 |
LMNA10F/LMNA10R |
ACC CTT CCC Tgg CCC TgA C |
CAC CTg ggT TCC CTg TTC AAg |
|
|
|
|
|
|
10B |
LA10FB/LA10RB |
gCA AgC Tgg TgC gCT Cag Tg |
ggC CAg gCC AgC gAg TAA Ag |
|
|
|
|
|
|
11 |
LMNA11F/LMNA11Rev |
ggT CAg TCC CAg ACT CgC C |
CgC CTg CAg gAT TTg gAA gAC |
|
|
|
|
|
|
12 |
LMNA12F/LMNA11R |
gTg Agg gAT ggg ggA gAT gC |
ggg Tgg gCA TgA ggT gAg g |
|
EXON |
Temperature of analysis °C |
Gradient of acetonitrile (%) |
|---|---|---|
|
1A |
66 |
51-56-64 |
|
|
68 |
49-54-62 |
|
1B |
65 |
52-57-65 |
|
|
66 |
51-56-64 |
|
2 |
62 |
51-56-64 |
|
|
64 |
49-54-62 |
|
3 |
61 |
48-53-61 |
|
|
63 |
46-51-59 |
|
4B |
63 |
50-55-63 |
|
|
66 |
45-50-58 |
|
5 |
65 |
48-53-61 |
|
6 |
65 |
50-55-63 |
|
7 |
64 |
52-57-65 |
|
|
65 |
51-56-64 |
|
8 |
62 |
48-53-61 |
|
|
64 |
45-50-58 |
|
9 |
64 |
49-54-63 |
|
10 |
65 |
48-53-61 |
|
10B |
64 |
46-51-59 |
|
|
66 |
45-50-58 |
|
11 |
65 |
52-57-65 |
|
|
67 |
48-53-61 |
|
12 |
62 |
47-52-60 |
|
|
63 |
46-51-59 |
|
EXON |
primers |
HOT START |
DENAT |
ANNEAL |
ELONG |
fin. ext. |
n°cycles |
Cycler |
|---|---|---|---|---|---|---|---|---|
|
1 |
1NF/1R |
96° 5 ' |
96°30'' |
64°45'' |
72°45'' |
72° 7' |
40x |
9600 |
|
|
|
|
|
|
|
|
|
|
|
2 |
2F/2NR |
96° 5 ' |
96° 30'' |
61° 30'' |
72° 30'' |
72° 7' |
40x |
9700 |
|
|
|
|
|
|
|
|
|
|
|
3 |
3NF/3NR |
96° 5 ' |
96° 30' |
63° 30'' |
72° 30'' |
72° 7' |
35 |
9700 |
|
|
|
|
|
|
|
|
|
|
|
4 |
4F/4R |
96° 5' |
96° 1' |
66°1' |
72° 1' |
72° 7' |
40x |
9600 |
|
|
|
|
|
|
|
|
|
|
|
5 |
5AF/6AR |
96° 5' |
96° 1' |
67°1' |
72° 1' |
72° 7' |
40x |
9700 |
|
|
|
|
|
|
|
|
|
|
|
6A |
6AF/6AR |
96° 5' |
96° 30' |
64° 30'' |
72° 30'' |
72° 7' |
35 |
9700 |
|
|
|
|
|
|
|
|
|
|
|
6B |
6BF/6NBR |
96° 5' |
96° 1' |
61°1' |
72° 1' |
72° 7' |
40x |
9600 |
|
|
Forward |
Reverse |
|---|---|---|
|
|
|
|
|
1NF/1R |
GCCGCTCCCAATGCGCTCG |
CCCTCCTCGTGCTCCGCCT |
|
|
|
|
|
2F/2NR |
TCGTGCTCCGCCTCGCGAC |
GGATGGGGTCGCGAGGGTG |
|
|
|
|
|
3NF/3NR |
GGTCGAGAGCGGCACTGGAG |
GACCCCGCTCACAGGGAGGA |
|
|
|
|
|
4F/4R |
CAGGCGGGGCACACCGATGC |
CCTAGGGGATCGCGGCTGTGT |
|
|
|
|
|
5AF/6AR |
CCCTGCCAGCCAGTCCCCTC |
CATGGCTCACCCGCCGTGCC |
|
|
|
|
|
6AF/6AR |
CCAGGGACGGGCTGGTTCTG |
CATGGCTCACCCGCCGTGCC |
|
|
|
|
|
6BF/6NBR |
AGAGCATCACGCACTACCGC |
CTTCAGAGCTAGGTCTTTGGGG |
|
Exon |
Analysis temperature |
Gradient B [%] |
|---|---|---|
|
1 |
67 |
48-53-61 |
|
|
69 |
47-52-69 |
|
|
|
|
|
2 |
64 |
50-55-63 |
|
|
66 |
48-53-61 |
|
|
|
|
|
3 |
60 |
45-50-58 |
|
|
61 |
47-52-60 |
|
|
|
|
|
4 |
63 |
48-53-61 |
|
|
64 |
47-52-60 |
|
|
|
|
|
5 |
60 |
51-56-64 |
|
|
61 |
51-56-64 |
|
|
|
|
|
6A |
64 |
44-49-67 |
|
|
|
|
|
6B |
62 |
49-54-62 |
|
|
65 |
43-48-56 |
The Toniolo Lab at San Raffaele, Home page | The Toniolo Lab at San Raffaele, 'supplementary Data' page