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Mlr启动子合成与验证

Synthesis and Characterisation of the Mlr Promoter

Research background

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086–4094). And the gene cluster involved in bacterial degradation of the cyanobacterial
toxin microcystin LR were be reported by David G. Bourne in 2001. The cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the “penicillin-binding enzyme” family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.
In this study, we synthesized a RFP-promoter-GFP sequence to detect the relationship between MLR promoter and microcystin LR. This synthesized sequence using the promoter sequence between mlrA and mlrC, adding the red fluorescent protein sequence in the upstream to instead mlrC gene, and adding green fluorescent protein sequence in the downstream to instead mlrA gene. cvv

Fig 1 Restriction map of the 5.8 kb of sequence data and localisation of genes involved in microcystin LR degradation.

methods

Synthetic sequence

BamHI
GCCTGCAGctataaaaataaatgatgtctaccttctgttctttcatattgttcaacaattgtataatcttcattatgtgatgtaatatctaacttagcatctacataataatatcctggtaattgaactggttttttagccatataaattgatttaaattcaactaaataatgtcctccgtcctttaattttagagctttatgtgtttctccctttaaaaccccgtctcttggatataatctttctgtactagcttcccatcccattgtttttttttgcataactggtccgtctgatggaaaattaaccccaataaatttaactttataaataaaacaaccatcttgtaatgatgaatcttgtgtaactgtagcaactccaccatcttcaaaattcataactctttcccatttaaatccttctggaaaagataattttttataatctggaatatcagctggatgtttaacgtaaacctttgatccatattgaaattgtggtgataaaatatcccacgcaaatggtaatggtcctccttttgtaacctttaatttaactgtattatgtccttcataCggtctaccttctccttctccctcaatttcaaattcatgtccattaactgtaccttccattctaactttaaatctcataaattctgtaataacattttctgatgaagccatAacacgagtcttcggtttgctgttgtttgcaccagctgctgcatcttcaccccataaatcaaccgaacggacagtcaattcttgttgaccatcctgggcctatctgaaatgttctgctgacagaacgggagaattgaaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcaCatgaaacagcatgactttttcaagagtgccatgcctgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaGAATTCCG
EcoRI

Sequence analysis

_Base Count : 1551 bp (489 A, 496 T, 314 C, 252 G)
_Composition : 36% GC, 64% AT

Absent Sites
AarI AatII Acc65I AciI AclI AfeI AflII AgeI AhdI AleI ApaI ApaLI AscI AseI AsiSI AvaI BamHI BanI BanII BbvCI BceAI BciVI BclI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BsaBI BsaHI BsaWI BseMII BseRI BsgI BsiEI BsiHKAI BsiWI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI BsrDI BsrFI BssHII BstEII BstUI Bsu36I BtrI BtsI Cac8I ClaI DraIII EagI EarI EciI Eco53KI EcoNI EcoO109I EcoRV FauI FseI FspAI FspI HaeII HgaI HhaI HinP1I HindIII HpaII Hpy99I KasI KpnI MluI MmeI NaeI NarI NciI NdeI NgoMIV NheI NlaIV NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PfoI PmeI PpuMI PshAI PspOMI PspXI PstI PvuI RsrII SacI SacII SalI SanDI SbfI ScaI SexAI SfiI SfoI SgrAI SmaI SnaBI SpeI SphI SrfI SspI StuI TauI TspMI Tth111I XbaI XcmI XhoI XmaI XmnI ZraI
Unique Sites
Unique Sites
BamHI(3)
DdeI (92)
Esp3I (223)
SfcI (367)
BstXI (512) MslI (513)
BssSI (688) PleI (691) BbsI (693)
MwoI (714)
BpmI (851)
BsmFI (859)
Bsp1286I (895)
BtgI (991) MscI (994) Tsp45I (1006)
BsrGI (1101)
AflIII (1147) BsaAI (1148)
BstZ17I (1273)
HpaI (1312)
BtgZI (1391)
AsuII (1446) BstYI (1452) BsaI (1469) DrdI (1471)
SmlI (1486) BseYI (1504)
EcoRI (1543)

Building Block Design

The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature.

2 building blocks were generated.
Building Block mlrAC.1 789bp 1..789
Left - 5' GCCCTAGGCTATAAAAATAAATGATG 3'
Rght - 5' ATAGGCCCAGGATGGTCAA 3'
RghtU - 5' ATAGGCCCAGGAUGGTCAA 3'
Sequence:
Assembly Oligos: average overlap Tm is 48°;average oligo length is 56bp.

GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAATTGTATAATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAATTGAACTGGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCCTTTAATTTTAGAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTACTAGCTTCCCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCCCAATAAATTTAACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCAACTCCACCATCTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAATCTGGAATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAAATATCCCACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTCATACGGTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTTCCATTCTAACTTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGAGTCTTCGGTTTGCTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGACAGTCAATTCTTGTTGACCATCCTGGGCCTAT
GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA
AGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTG GTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATT TTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGG AGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACG GCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACAT ACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGG GGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGC TAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGG GAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGG AGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGT GACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACG GGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA
CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGAT

CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATTTAAGTTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGGAAATTTTGGGGCAGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACGTATTGACCAGGCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACATTACTACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGGTAAATTTAGGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGCATTTGGAAACTAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGGAGGAAAACATTGGAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGGAGTTAAAGTTTAAGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGTAAAAGACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACGACGTAGAAGTGGGGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA    .
Building Block mlrAC.2   775bp   777..1551
Left - 5' ATCCTGGGCCTATCTGAAATG 3'
LeftU - 5' ATCCTGGGCCTAUCTGAAATG 3'
Rght - 5' CGGAATTCTTATTTGTATAGTTCATCC 3'
Sequence:
Assembly Oligos: average overlap Tm is 49°;average oligo length is 53bp.
ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCTGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGAATTCCG
ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCAT            AGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG       TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT          GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG            ATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGC          GGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA           GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT     AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA     TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTG     GGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGG         TTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCC           GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATG
                                CTTGCCCTCTTAACTTGGTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAG          AACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCC            TTGTATGCCTTTTGAATGGGAATTTAAATAAACGTGATGACCTTTTGATGGA            TGTGAACAGTGATGAAAGCCAATACCACAAGTTACGAAACGCTCTATGGGTC            TCGTACTGAAAAAGTTCTCACGGTACGGACTTCCAATACATGTCCTTTCTTGAT          TCTACTGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGG          CTTAGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTG         ATGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACC         ATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCT           TTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGAC         AGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAA           TCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATTCTTAAGGC
TAGGACCCGGATAGACTTTACAAGACGACTGTCTTGCCCTCTTAACTTGGTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCACTTCCACTACGTTGTATGCCTTTTGAATGGGAATTTAAATAAACGTGATGACCTTTTGATGGACAAGGTACCGGTTGTGAACAGTGATGAAAGCCAATACCACAAGTTACGAAACGCTCTATGGGTCTAGTGTACTTTGTCGTACTGAAAAAGTTCTCACGGTACGGACTTCCAATACATGTCCTTTCTTGATATAAAAAGTTTCTACTGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGGGAACAATTATCTTAGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTGTTTAACCTTATGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACCTTAGTTTCAATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCTGGTAATAGTTGTTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGACAGGTGTGTTAGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAACTCAAACATTGTCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATTCTTAAGGC

Primer synthesis

Primer synthesis order o Comate Bioscience Co.,Ltd.
吉林省库美生物科技有限公司引物合成订购单

引物编号	序列，(5' to 3')	碱基数（bp）	合成总量(OD)	分装管数	纯化方式
AC101	GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT	59	1	1	PAGE
AC102	GTTAGATATTACATCACATAATGAAGATTATACAATTGTTGAACAATATGAAAGAACAGA	60	1	1	PAGE
AC103	ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT	58	1	1	PAGE
AC104	TTAAATCAATTTATATGGCTAAAAAACCAGTTCAATTACCAGGATATTATTATGTAGATG	60	1	1	PAGE
AC105	GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC	54	1	1	PAGE
AC106	GGGAGAAACACATAAAGCTCTAAAATTAAAGGACGGAGGACATTATTTAGTT	52	1	1	PAGE
AC107	GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC	57	1	1	PAGE
AC108	GCAAAAAAAAACAATGGGATGGGAAGCTAGTACAGAAAGATTATATCCAAGAGA	54	1	1	PAGE
AC109	CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC	53	1	1	PAGE
AC110	TACAAGATGGTTGTTTTATTTATAAAGTTAAATTTATTGGGGTTAATTTTCCATCAGACG	60	1	1	PAGE
AC111	ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA	54	1	1	PAGE
AC112	GGGAAAGAGTTATGAATTTTGAAGATGGTGGAGTTGCTACAGTTACACAAGATTCA	56	1	1	PAGE
AC113	CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT	60	1	1	PAGE
AC114	CGTTAAACATCCAGCTGATATTCCAGATTATAAAAAATTATCTTTTCCAGAAGG	54	1	1	PAGE
AC115	AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA	58	1	1	PAGE
AC116	GGACCATTACCATTTGCGTGGGATATTTTATCACCACAATTTCAATATGGAT	52	1	1	PAGE
AC117	CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC	59	1	1	PAGE
AC118	GGGAGAAGGAGAAGGTAGACCGTATGAAGGACATAATACAGTTAAATTAAAG	52	1	1	PAGE
AC119	GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT	54	1	1	PAGE
AC120	TGTTATTACAGAATTTATGAGATTTAAAGTTAGAATGGAAGGTACAGTTAATGGACATGA	60	1	1	PAGE
AC121	TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA	52	1	1	PAGE
AC122	GCAGCTGGTGCAAACAACAGCAAACCGAAGACTCGTGTTATGGCTTCATCAG	52	1	1	PAGE
AC123	CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA	52	1	1	PAGE
AC124	ATAGGCCCAGGATGGTCAACAAGAATTGACTGTCCGTTCGGTTGATTTATGG	52	1	1	PAGE
AC201	ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCAT	52	1	1	PAGE
AC202	GACAACTCCAGTGAAAAGTTCTTCTCCTTTACTCATGGTTCAATTCTCCCGTTC	54	1	1	PAGE
AC203	AGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG	57	1	1	PAGE
AC204	CCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAA	52	1	1	PAGE
AC205	TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT	54	1	1	PAGE
AC206	AGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTT	52	1	1	PAGE
AC207	GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG	52	1	1	PAGE
AC208	CTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGT	52	1	1	PAGE
AC209	ATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGC	54	1	1	PAGE
AC210	TAGTTCTTTCCTGTACATAACCTTCAGGCATGGCACTCTTGAAAAAGTCATGCT	54	1	1	PAGE
AC211	GGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA	52	1	1	PAGE
AC212	GGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCT	52	1	1	PAGE
AC213	GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT	57	1	1	PAGE
AC214	GTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTC	53	1	1	PAGE
AC215	AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA	57	1	1	PAGE

AC215	AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA	57	1	1	PAGE
AC216	CCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTA	53	1	1	PAGE
AC217	TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTG	57	1	1	PAGE
AC218	TCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTA	51	1	1	PAGE
AC219	GGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGG	53	1	1	PAGE
AC220	CAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTT	53	1	1	PAGE
AC221	TTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCC	51	1	1	PAGE
AC222	AAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGA	51	1	1	PAGE
AC223	GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATG	51	1	1	PAGE
AC224	CGGAATTCTTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGCAGCT	51	1	1	PAGE
AC1	GCCCTAGGCTATAAAAATAAATGATG	26	1	1	PAGE
AC2	ATAGGCCCAGGATGGTCAA	19	1	1	PAGE
AC3	ATCCTGGGCCTATCTGAAATG	21	1	1	PAGE
AC4	CGGAATTCTTATTTGTATAGTTCATCC	27	1	1	PAGE

二、实验操作及结果分析

1、引物溶解

根据合成引物的分子量，计算需要添加的灭菌去离子水的量，将合成的引物稀释成10um/L备用。

2、引物混合(只有表格不一样，文字叙述是一样的)

合成的寡核苷酸呈粉末状，使用前需要10000rpm离心1min，使寡核苷酸干粉集中在管底，使用时小心开启瓶盖。
溶解寡核苷酸前核对合成报告单与引物标签上的OD数是否一致，并核对分装管数。根据每个引物的合成报告单计算出配制10umol/L浓度的寡核苷酸需要加入水的体积，加入去离子无菌水，室温放置2min，反复振荡助溶，再离心将溶液收集到管底。
每6条引物为一组，首末位引物取4ul，中间引物取1ul，加入新的EP管中，再补充灭菌去离子水至20ul，混匀备用。

组别	引物编号	吸取量	灭菌去离子水添加量	总体积	每PCR（50ul体系）吸取量	终浓度
A1	AC101	4	8	20	5ul	200nM
	AC102	1				50nM
	AC103	1				50nM
	AC104	1				50nM
	AC105	1				50nM
	AC106	4				200nM
A2	AC107	4	8	20	5ul	200nM
	AC108	1				50nM
	AC109	1				50nM
	AC110	1				50nM
	AC111	1				50nM
	AC112	4				200nM
A3	AC113	4	8	20	5ul	200nM
	AC114	1				50nM
	AC115	1				50nM
	AC116	1				50nM
	AC117	1				50nM
	AC118	4				200nM
A4	AC119	4	8	20	5ul	200nM
	AC120	1				50nM
	AC121	1				50nM
	AC122	1				50nM
	AC123	1				50nM
	AC124	4				200nM
A5	AC201	4	8	20	5ul	200nM
	AC202	1				50nM
	AC203	1				50nM
	AC204	1				50nM
	AC205	1				50nM
	AC206	4				200nM
A6	AC207	4	8	20	5ul	200nM
	AC208	1				50nM
	AC209	1				50nM
	AC210	1				50nM
	AC211	1				50nM
	AC212	4				200nM
A7	AC213	4	8	20	5ul	200nM
	AC214	1				50nM
	AC215	1				50nM
	AC216	1				50nM
	AC217	1				50nM
	AC218	4				200nM
A8	AC219	4	8	20	5ul	200nM
	AC220	1				50nM
	AC221	1				50nM
	AC222	1				50nM
	AC223	1				50nM
	AC224	4				200nM

Team:Jilin China/NOTEBOOK