心肌醛固酮合酶PCR
丁香园论坛
1377
由那位老师作过醛固酮合酶的RT-PCR，我已经P了两个月了，还是为扩出来。我用Wistar 大鼠的心肌及肾上腺组织提取RNA并RT，后进行PCR，已经换了3个引物，其中由两对引物是文献（英文一篇）报道的，但还未P出来，PCR条件我都试了好多次了，同样的标本P其他的3对基因都很顺利，就这对出不来，郁闷死了！！如果出不来结果以前的试验就白做了。下面是我的一对引物及基因序列：请帮忙分析一下，尤其是作过醛固酮合酶CYP11b PCR的老师请伸出援助之手！谢谢！！
up 5' ACCATGGATGTCCAGCAA 3'
down 5' CTCTCGACGGCTCAGACT 3'
1 gccgcttttt tttttttttt tttttaggta aatagtaaag cctttatttt cttgttaaga
61 acagcatttt gaaaataaaa cctatctgcc catgcttaac aacctttaaa gtctgtgata
121 ttttatatac agccctgtac atactgattg tctggaaatt tcttaaacag tttttgttta
181 taagtatgca agtcagccag gatgagggga agagtgaggg tacattataa aatacacatt
241 aatacattta ataaatatat attatctatc aaaaacgagc catagctctt aatgaataaa
301 gcacctgcca aggctctcag ggtgacagca gatgtgtggc tggcaagacc ctggcagtgc
361 ctgcacagga cgagggcact gggtactacg gcaacactgg cccctaagac actgaagccc
421 tttgaagcca taccacaata ctccaggaac aagtggctga agatgataca gatcctgagg
481 gagcagggcc aagagaacct acacctggag atgcaccagg ccttccagga gctggggccc
541 attttcaggc acagtgcagg gggagcacag attgtgtctg tgatgctgcc tgaggacgct
601 gagaagctgc accaggtgga gagtatcctc ccgcgtcgga tgcacctgga gccgtgggtg
661 gcccacaggg aactccgtgg cctgagacgt ggtgtgttct tgctaaatgg ggcagactgg
721 cgcttcaacc gactgaaact gaacccaaac gtgctgtcac caaaagctgt tcaaaatttt
781 gtccccatgg tggacgaggt agcaagggac ttcttggagg ccctgaaaaa gaaggtgcgt
841 cagaatgctc gagggagcct taccatggat gtccagcaaa gtctcttcaa ctacactata
901 gaagccagca actttgcact ttttggagag aggctgggcc tccttggtca tgacctgaac
961 cctggtagcc tgaagttcat ccatgcccta cattcaatgt tcaagtccac cacacagctc
1021 ctgttcttac ccagaagctt gactcgctgg acaagcactc gggtgtggaa agaacatttt
1081 gatgcctggg atgtcatctc tgagtatgcc aacagatgta tctggaaggt gcaccaggaa
1141 ctcagactcg gcagctctca gacctacagt ggcattgtgg cagcactaat aactcaggga
1201 gctttacctc tggacgccat caaagccaac tctatggagc tcactgctgg gagcgttgac
1261 acgacagcaa tccccttggt aatgaccctt tttgagctgg ctcggaaccc agatgttcag
1321 caggccctgc ggcaggagac cctggcagct gaggccagca tcgctgctaa tccccagaag
1381 gccatgtcag acctgccctt gctgcgggct gcccttaaag agaccttgag actctaccct
1441 gttggtggct ttttggagag aatcctaaac tcagacctgg tgcttcagaa ctatcatgtc
1501 cctgctggga cgttggtcct actttatctg tactccatgg gccgaaaccc tgcagtgttc
1561 ccaagacctg agcgctatat gcctcagcgc tggctggaga ggaaaaggag tttccagcat
1621 ctggccttcg gctttggggt gcgccagtgc ctggggcggc gcctggcaga ggtggagatg
1681 ctgctcctgc ttcaccatat gctgaaaacc ttccaggtgg agacactgag acaagaggat
1741 gtgcagatgg cctatcgctt tgttttgatg cccagctcta gtcctgtcct cactttccgg
1801 cccgtcagct agtcacctta catctggggc cctaatcagt ccaccggcct acttctgtcc
1861 tgacgcccaa ggtcacgctt ctctgccagc agaaatcttg gggggggggg aagaggtcac
1921 attgtcatca ttcaacacaa ctgtacacag tttacagctg aagacctcca gcatcaggta
1981 caagcaaggc cagggaagag attaggagta tcttcagggc aataggtagt ctctcctgag
2041 cggtcttccc cattctttcc aacggtcact ccaggtggcc acggccttca aagggtctca
2101 gagtaaagcc agaagtaatc tcctccttat ggatccatta ttttgacatg acccacaaaa
2161 acattttcac acacagatgg gtttttgggg ggcaaaagat acatagatga tagatatgat
2221 atatgatatt gatagataga tagatgatag atagatagat agatagatag atagatagat
2281 agatagatag atagatagat gatggatgac cagtcattta tgtctgtgtt ttgtttgaag
2341 cctttcacag tggagctggc acatatgagt tggtgactct acagtgtttt aagagtagac
2401 acagctgcac ttgctaagga ctgggtggtt gtggtatcag agatgcaggc aggaggacct
2461 gctggaaccc agatactgtg tgaagtcccc acagggacgg tggtcttcca tgagatcaag
2521 gtgttttcct gacttctgcc agtgcatgct gcttgggcaa aggtaattct cttgacttgt
2581 gaaagtggcc caaagcataa gaattttttc aggtatcctt tcagctgcaa gtcggtaggg
2641 cgaaaagttt tggccaagaa caaccacttc acaggatctc ctgtgctcag caccaaagca
2701 caaatctact ggagatgtgt gtgccaaatt ccaaggtctt aggcctttga gtgtgaggca
2761 gtatagagtc agggccttta atgatataat tcgtgtgaaa tgagattact gggttgtgcc
2821 tactccaatg cccagagagc acccaagaag agagtgaggt gccaggtcac agagacagac
2881 cccatgtgga agacaggaag tcagacttct gtaagccgag gaaagcagtt ggaactcctg
2941 tagtcttggc tccatcactc aatcacttcc ctggctgtga aaaataaact tgtgttgttg
up 5' ACCATGGATGTCCAGCAA 3'
down 5' CTCTCGACGGCTCAGACT 3'
1 gccgcttttt tttttttttt tttttaggta aatagtaaag cctttatttt cttgttaaga
61 acagcatttt gaaaataaaa cctatctgcc catgcttaac aacctttaaa gtctgtgata
121 ttttatatac agccctgtac atactgattg tctggaaatt tcttaaacag tttttgttta
181 taagtatgca agtcagccag gatgagggga agagtgaggg tacattataa aatacacatt
241 aatacattta ataaatatat attatctatc aaaaacgagc catagctctt aatgaataaa
301 gcacctgcca aggctctcag ggtgacagca gatgtgtggc tggcaagacc ctggcagtgc
361 ctgcacagga cgagggcact gggtactacg gcaacactgg cccctaagac actgaagccc
421 tttgaagcca taccacaata ctccaggaac aagtggctga agatgataca gatcctgagg
481 gagcagggcc aagagaacct acacctggag atgcaccagg ccttccagga gctggggccc
541 attttcaggc acagtgcagg gggagcacag attgtgtctg tgatgctgcc tgaggacgct
601 gagaagctgc accaggtgga gagtatcctc ccgcgtcgga tgcacctgga gccgtgggtg
661 gcccacaggg aactccgtgg cctgagacgt ggtgtgttct tgctaaatgg ggcagactgg
721 cgcttcaacc gactgaaact gaacccaaac gtgctgtcac caaaagctgt tcaaaatttt
781 gtccccatgg tggacgaggt agcaagggac ttcttggagg ccctgaaaaa gaaggtgcgt
841 cagaatgctc gagggagcct taccatggat gtccagcaaa gtctcttcaa ctacactata
901 gaagccagca actttgcact ttttggagag aggctgggcc tccttggtca tgacctgaac
961 cctggtagcc tgaagttcat ccatgcccta cattcaatgt tcaagtccac cacacagctc
1021 ctgttcttac ccagaagctt gactcgctgg acaagcactc gggtgtggaa agaacatttt
1081 gatgcctggg atgtcatctc tgagtatgcc aacagatgta tctggaaggt gcaccaggaa
1141 ctcagactcg gcagctctca gacctacagt ggcattgtgg cagcactaat aactcaggga
1201 gctttacctc tggacgccat caaagccaac tctatggagc tcactgctgg gagcgttgac
1261 acgacagcaa tccccttggt aatgaccctt tttgagctgg ctcggaaccc agatgttcag
1321 caggccctgc ggcaggagac cctggcagct gaggccagca tcgctgctaa tccccagaag
1381 gccatgtcag acctgccctt gctgcgggct gcccttaaag agaccttgag actctaccct
1441 gttggtggct ttttggagag aatcctaaac tcagacctgg tgcttcagaa ctatcatgtc
1501 cctgctggga cgttggtcct actttatctg tactccatgg gccgaaaccc tgcagtgttc
1561 ccaagacctg agcgctatat gcctcagcgc tggctggaga ggaaaaggag tttccagcat
1621 ctggccttcg gctttggggt gcgccagtgc ctggggcggc gcctggcaga ggtggagatg
1681 ctgctcctgc ttcaccatat gctgaaaacc ttccaggtgg agacactgag acaagaggat
1741 gtgcagatgg cctatcgctt tgttttgatg cccagctcta gtcctgtcct cactttccgg
1801 cccgtcagct agtcacctta catctggggc cctaatcagt ccaccggcct acttctgtcc
1861 tgacgcccaa ggtcacgctt ctctgccagc agaaatcttg gggggggggg aagaggtcac
1921 attgtcatca ttcaacacaa ctgtacacag tttacagctg aagacctcca gcatcaggta
1981 caagcaaggc cagggaagag attaggagta tcttcagggc aataggtagt ctctcctgag
2041 cggtcttccc cattctttcc aacggtcact ccaggtggcc acggccttca aagggtctca
2101 gagtaaagcc agaagtaatc tcctccttat ggatccatta ttttgacatg acccacaaaa
2161 acattttcac acacagatgg gtttttgggg ggcaaaagat acatagatga tagatatgat
2221 atatgatatt gatagataga tagatgatag atagatagat agatagatag atagatagat
2281 agatagatag atagatagat gatggatgac cagtcattta tgtctgtgtt ttgtttgaag
2341 cctttcacag tggagctggc acatatgagt tggtgactct acagtgtttt aagagtagac
2401 acagctgcac ttgctaagga ctgggtggtt gtggtatcag agatgcaggc aggaggacct
2461 gctggaaccc agatactgtg tgaagtcccc acagggacgg tggtcttcca tgagatcaag
2521 gtgttttcct gacttctgcc agtgcatgct gcttgggcaa aggtaattct cttgacttgt
2581 gaaagtggcc caaagcataa gaattttttc aggtatcctt tcagctgcaa gtcggtaggg
2641 cgaaaagttt tggccaagaa caaccacttc acaggatctc ctgtgctcag caccaaagca
2701 caaatctact ggagatgtgt gtgccaaatt ccaaggtctt aggcctttga gtgtgaggca
2761 gtatagagtc agggccttta atgatataat tcgtgtgaaa tgagattact gggttgtgcc
2821 tactccaatg cccagagagc acccaagaag agagtgaggt gccaggtcac agagacagac
2881 cccatgtgga agacaggaag tcagacttct gtaagccgag gaaagcagtt ggaactcctg
2941 tagtcttggc tccatcactc aatcacttcc ctggctgtga aaaataaact tgtgttgttg
