Mut = CCC-
WT= CCCC
AACCTCCACGACTAAATGCATGATAAATACTTTAGTTAAGTCATGAATGCATGAAGTGT
ATGTGTTGGTGTATGACTGGGGATTTAAGGAGCTAACAGGGAATGGATATAAGCAGAG
GGAATGACACACATCTGTGAAGATAAAGATGTCACAGTGAGACCCTAGTTTGTATATA
CATTAAACATAGTTTTAAAGAGGGCCTGGGGATGAGGCTGAGACACTGAAGTGCCAG
GAGGGCAATGGAACCTGAGATTGATCGACCGGAACCCACGTGAACTATCTGGACGCA
GTGGGGCATACTTAAGATACCAAATTTGGAAAGGGAGAGAGACAGGGGTCCTTGAGT
TTTGCTGGATATCCAGTTTCCCCCAACCAGGAAACCCCAGGCTCAGTGAAAGATCCT
GAATCAAAAAACTAAAGTGTATAGAGGAAAACACCTGATATTGAGCTCTGACCTGTA
CGCATACACCCACCACATTAAATAAATAAATAAATAAATAAATAAATAAATAAATAAAT
AAATAAAATACACACATACACATGCATGCACATGCATACACATACATACATATACATAC
ATATAAATACATATACATACATACTTATTTACTTACTTACTTAAGACAAGTTAGAAAGTA
ATGAGACATAGTAATGCATTATATATACAATCCTAAGTCATTGCCTTAGGATTTGTTGAT
CAAGGGAGGCTACTGGGGGAAAGAAGTCTTTCTCACCTCAAATTTTATCTTCCAGGA
TTT(cccc)ATACCTTGATGTGTTCCCTCAATAATACATCTTTGGTCCCCATGACCTTCAAA
CTGCGTGTCCGCGGCGATGGCGAAGGCATGAGTAGCATCCCAAGTTACTCTCAGGAG
TCAGACAGCAAGCAATGGTCCGGGATTAATACAGAAATGCCTACCACAAAACCCAAA
GAATTCACCATCTCTCCCAACAGTGGCACCATCCGTGCCCAGGGCTTTACTGCCATCAAGGTAAAGTGT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
25792 | TGA TCA AGG GAG GCT ACT GG | Forward | A | |||
25793 | ACC ATT GCT TGC TGT CTG ACT | Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
25792 | 0.50 uM |
25793 | 0.50 uM |
Glycerol | 6.50 % |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -1.0 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles | |
6 | 94.0 | -- | |
7 | 55.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |