Thermal Asymmetric Interlaced Tail PCR
From： 绿谷生物网 Update： 2008-02-12
TAIL1 TAIL2
Cycle # Temp (Celsius) Time Cycle # Temp (Celsius) Time
1 95 2 min 1 95 2 min
2 94 30 sec 2 94 10 sec
3 67 1 min 3 64 1 min
4 72 2:30 4 72 2:30
5 Go to 2 4 times 5 94 10 sec
6 94 30 sec 6 64 1 min
7 25 3 min 7 72 2:30
8 25-72 @0.3 degrees/sec 8 94 10 sec
9 72 2:30 9 44 1 min
10 94 10 sec 10 72 2:30
11 67 1 min 11 Go to 2 11 times
12 72 2:30 12 72 5 min
13 94 10 sec 13 4 forever
14 67 1 min
15 72 2:30
16 94 10 sec
17 44 1 min
18 72 2:30
19 Go to 10 14 times
20 72 5 min
21 4 forever
Primer sequences: The 10 pmol/ m l stock is 100x for the TIR primers and 10x for the AP's. TIR6 is used in TAIL1. The four TIR8 primers are mixed together in proportions normalizing their degeneracy (an 11 m l mixture would have 2, 4, 4, 1 m l, respectively of the primers)
Primer Sequence Length #GC %GC Tm Degeneracy
TIR6 AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC 32 17.5 54.7 71.7 4
TIR8.1 CGCCTCCATTTCGTCGAATCCCCTS 25 15 60.0 69.5 2
TIR8.2 CGCCTCCATTTCGTCGAATCCSCTT 25 14 56.0 67.9 4
TIR8.3 SGCCTCCATTTCGTCGAATCCCKT 25 14.5 58.0 68.7 4
TIR8.4 CGCCTCCATTTCGTCGAATCACCTC 25 14 56.0 67.9 1
Tm=81.5 + (16.6 x log[Na+]) + (41x (#G+#C)/ #bases) - 500/#bases
These are the original primers developed by Liu, et al (1995) for Arabidopsis We sometimes use these, but SAD11 and SW41 will cover most of what they would hit :
AD1 XTCGASTWTSGWGTT
AD2 XGTCGASWGAXAWGAA
AD3 WGTGXAGWAXCAXAGA
W4 AGWGXAGWAXCAXAGA
These are the new primers developed by our group that are optimized for maize:
Primer Sequence Length #GC %GC Tm Notes/Degeneracy
cggc1 GVCTYCGWSSGC 12 9.16 76.33 54.5 48
SAD11 NTCAGSTWTSGWGWT 15 6.50 43.33 49.3 AD1 deg 128
SW41 AGWGHAGSAHCADAAS 16 7.00 43.75 51.6 W4 no cp seq/216
BAD5 WTCCASNTGSNACG 14 7 50.00 49.7 128
DRM-CG2 GCNGNWCGWCGWG 13 8.5 65.38 53.2 128
CST1 GTANTCGWAWNCST 14 5.5 39.29 45.3 128
CTG1 GWWGGTSCWASWCTG 15 8 53.33 53.4 64
AMS2=GAG3 GWSIDRAMSCTGCTC 15 8.3 55.33 54.3 96
geeky1 GKYKGCKGCNGC 12 9.5 79.17 55.7 64
DRM-AG1 GNGWSASTNGAGC 13 7.5 57.69 50.1 128
BAD8 GTGASNTGSWATGG 14 7 50.00 49.7 32
DRM-NC1 GSCNCSGWNCC 11 8.5 77.27 51.1 128
Protocol developed by Sue Latshaw, Mark Settles, Bao-Cai Tan and Don McCarty at the University of Florida.
