la Taq也没P出来,急！！急！！

我做大鼠心肌的醛固酮合酶RT－PCR，都2个月了，一点结果都没有，偶尔出一条带还不是我所需要的条带，最近有人看了我的基因序列发现局部GC 含量高，建议用la Taq,就又花了300多元反复作了多次还是没有结果，用脑及肾上腺的cDNA作阳性对照也同样，而用同样的cDNA能扩出其他4对目的基因。真郁闷啊，快过年了，老板说节前做不出来节后先上班，业余时间作试验，一定要做出来。看来春节过不好了！那位高手能指点迷津,在下多谢了。
我的条件是94度2min预变性，94度30se,58-68度30－40se（都试过1次）,72度1min,35－40循环，用的Takara的lat aq,2xGC buffer
up 5' ACCATGGATGTCCAGCAA 3'
down: 5’ gagagctgccgagtctga 3'
CYP11b:
gccgcttttttttttttttttttttaggtaaatagtaaagcctttattttcttgttaaga
acagcattttgaaaataaaacctatctgcccatgcttaacaacctttaaagtctgtgata
ttttatatacagccctgtacatactgattgtctggaaatttcttaaacagtttttgttta
taagtatgcaagtcagccaggatgaggggaagagtgagggtacattataaaatacacatt
aatacatttaataaatatatattatctatcaaaaacgagccatagctcttaatgaataaa
gcacctgccaaggctctcagggtgacagcagatgtgtggctggcaagaccctggcagtgc
ctgcacaggacgagggcactgggtactacggcaacactggcccctaagacactgaagccc
tttgaagccataccacaatactccaggaacaagtggctgaagatgatacagatcctgagg
gagcagggccaagagaacctacacctggagatgcaccaggccttccaggagctggggccc
attttcaggcacagtgcagggggagcacagattgtgtctgtgatgctgcctgaggacgct
gagaagctgcaccaggtggagagtatcctcccgcgtcggatgcacctggagccgtgggtg
gcccacagggaactccgtggcctgagacgtggtgtgttcttgctaaatggggcagactgg
cgcttcaaccgactgaaactgaacccaaacgtgctgtcaccaaaagctgttcaaaatttt
gtccccatggtggacgaggtagcaagggacttcttggaggccctgaaaaagaaggtgcgt
cagaatgctcgagggagccttaccatggatgtccagcaaagtctcttcaactacactata
gaagccagcaactttgcactttttggagagaggctgggcctccttggtcatgacctgaac
cctggtagcctgaagttcatccatgccctacattcaatgttcaagtccaccacacagctc
ctgttcttacccagaagcttgactcgctggacaagcactcgggtgtggaaagaacatttt
gatgcctgggatgtcatctctgagtatgccaacagatgtatctggaaggtgcaccaggaa
ctcagactcggcagctctcagacctacagtggcattgtggcagcactaataactcaggga
gctttacctctggacgccatcaaagccaactctatggagctcactgctgggagcgttgac
acgacagcaatccccttggtaatgaccctttttgagctggctcggaacccagatgttcag
caggccctgcggcaggagaccctggcagctgaggccagcatcgctgctaatccccagaag
gccatgtcagacctgcccttgctgcgggctgcccttaaagagaccttgagactctaccct
gttggtggctttttggagagaatcctaaactcagacctggtgcttcagaactatcatgtc
cctgctgggacgttggtcctactttatctgtactccatgggccgaaaccctgcagtgttc
ccaagacctgagcgctatatgcctcagcgctggctggagaggaaaaggagtttccagcat
ctggccttcggctttggggtgcgccagtgcctggggcggcgcctggcagaggtggagatg
ctgctcctgcttcaccatatgctgaaaaccttccaggtggagacactgagacaagaggat
gtgcagatggcctatcgctttgttttgatgcccagctctagtcctgtcctcactttccgg
cccgtcagctagtcaccttacatctggggccctaatcagtccaccggcctacttctgtcc
tgacgcccaaggtcacgcttctctgccagcagaaatcttgggggggggggaagaggtcac
attgtcatcattcaacacaactgtacacagtttacagctgaagacctccagcatcaggta
caagcaaggccagggaagagattaggagtatcttcagggcaataggtagtctctcctgag
cggtcttccccattctttccaacggtcactccaggtggccacggccttcaaagggtctca
gagtaaagccagaagtaatctcctccttatggatccattattttgacatgacccacaaaa
acattttcacacacagatgggtttttggggggcaaaagatacatagatgatagatatgat
atatgatattgatagatagatagatgatagatagatagatagatagatagatagatagat
agatagatagatagatagatgatggatgaccagtcatttatgtctgtgttttgtttgaag
cctttcacagtggagctggcacatatgagttggtgactctacagtgttttaagagtagac
acagctgcacttgctaaggactgggtggttgtggtatcagagatgcaggcaggaggacct
gctggaacccagatactgtgtgaagtccccacagggacggtggtcttccatgagatcaag
gtgttttcctgacttctgccagtgcatgctgcttgggcaaaggtaattctcttgacttgt
gaaagtggcccaaagcataagaattttttcaggtatcctttcagctgcaagtcggtaggg
cgaaaagttttggccaagaacaaccacttcacaggatctcctgtgctcagcaccaaagca
caaatctactggagatgtgtgtgccaaattccaaggtcttaggcctttgagtgtgaggca
gtatagagtcagggcctttaatgatataattcgtgtgaaatgagattactgggttgtgcc
tactccaatgcccagagagcacccaagaagagagtgaggtgccaggtcacagagacagac
cccatgtggaagacaggaagtcagacttctgtaagccgaggaaagcagttggaactcctg
tagtcttggctccatcactcaatcacttccctggctgtgaaaaataaacttgtgt
上游引物位置861-879bp,下游1141-1159bp,产物长度 298bp