St ep 1.打开NCBI主页: 打开的页面如下：HarinnaJ 亡■n'wr iar ShlKhnDiogy irrfannBlkinWeJCDin± 1O NC<tCM KniM C<2|Tht HsMv O™rlw0<*MPOEOCcrewc SJU^TEi rwQwnKBJNiin-Ti・・FQ>E TL»iatia'airlKriOv>p hi-uiw^ an s«K*疋 SfQLWflS k* Bi悄XMriHVXi?BjnFl■土 kbHTT£+?9 hi BiptrHiia RTtw vvranAll !>«■■* E a 呼4^ 111口*ur PCRpmwa amdd-»Kriiin tartpK*3YHid h«Fiadmvj砂J乃如畑xmE ■町HIX■冲Ch中疋加RC0 仲需 is EW P W iL ”LtalAffiTALAI沏Ml■町IO戟桝f^iM-jrc*l-*4IEh・d・ I.M1- utHsnte Cff&f Hiu rTWirilEkrHO.4 E^nsicill4 Hid dm rAdMTfrrwra LTilanditWYWKgfSnrilVtotauJai 丄3临初Weicww to NCBI“F kF<«91ty 9WP! ■”i:aril-ih°ULWkiv kE-rw^-sd nd-^VKiiE rinn^aii出绅■WhE耳 翩i| MS#P"卜HbTsi加M HtWX^I R耳G^rWrtypt ^rid PfiirtfttyptCUA W 口"dir* WhU AliiMIblltfi ilLr3Hil IBdr IISL^ ^riiTiKI bld diMhlli. ±m iiictf viilifciai. praitKali. kicj ■ rjjp 口 i_IWmPljgE CE"W 5>*jiWIEWTCmFrc+in>]EC>CEiHTi^udC^fusna ■SWCMflFubChanH(W l&...Z«ai td Mkd al^jrtcfc如ZW'WfliiE■瞅■畑小51輯*«1 Pftliiifiri d him*耳|E! a c■科Am i 'i i*i3**i 3 pHmhM ai^iaiMO«啊PGRoffiM vn 阳*i^n> WMWiK*Hwd h bJnr*r-■ [aw ■■-pwwpriiduiMiaii unuriia ii'itoA tv?HWMt a*iM&>5rwvn.W-M..d利 1TOK.3WTHnn.rm»M HtW&MEZWIM 旳t»曲 41 T»K曲机町■土曲mbrZOgrta Swf-waHr^iMiAiaiu-^H-IKD Hiat a MdnkItZA Hbwt -„kb>ru " ■ *■.< W5ZVNn» ugud ：]]9 S3BI EltH 4：-B H・wv a 丹吋比 Ivlina阳xi=.得到如下页面:% NCBlEntrez GeneQtipfa^ Eumnv^- ▼ $!»■■ 2D by 厲曲"3»« ▼ Send ta *[缈 1* [ cunurtQrty 背[faawe jgmwwa; H J 和IP1 &3脑阳而ItfimE ：1 ・ 15 dF L5□ l!API rw»^iQmv； L1®.AHiatdtKWi: CtTOfnosirnE LG9( NC 期4FE.T 灯询出记了/144#刖耐 cnmptB-nmi i7*fl138□』：TTHERM OOTB&41O lwcseuAP1 VETALA.1 -MB IWS-IWc, JWI僧|阳附呼慢旺 航伸溯钏QlM* rn-ERM_QO139+10Gerumlc corrtext kacronirkarQamB TB3SB»4： L汕門ev^FI MEig.i •购 IW8-bo( iSo^urn 腋粗借创個|□軟 BgWTOMTO«Mmpew剧 I僧s wWa]L1KSunKi进一步获得该基因在NCBI里面的基因信息，到此我为什么要做这一步呢，主要是想获得该gene在拟南芥中的系统名，见下图:□ 1： AP1 AP1 (APETALA1); IDNIA binding I protein binding ? protein hEterodimerization^ transcription activatari1 transcripti-on factor [ Arabidopsts 茁訥mnm] 石电E斗丁244 updated d-JBrvKDICISummary ・?Ge tie s^mlbol APIGiene des-criptiiani API (APETALfil1); DNA binding f protein binding / protein heteradimerizati口口/ transerption a匚tivatar/ transerption factorPrimary source TA[R:ATIG^9JZLku-k tag AT 120苗娈运勺化取.拟G£Hte type protein coding由芥屮的杀轨名ReiSeq status REVIEWEDOrganism Arab^os^ tfiaiLineage E曲日rytr占日；Vra iT>s>ds; eurosfdsAlso knoWO OS AGAiM0U5-L[K；E吕打白 /e^otvoe. CnitfiTi^a 1 fip^ntae; St?即m閃f切 Brnfi■厂ZT; Brsssfca^s; firassicaceae.AGL7； API； ^ETAJLAl； F+h丁nsafi日ciMf苗f 申ZEQg閃y® Msgnoftrp^^td1； eutfiiccrtyfedt'ns-; ccne ■BGitfiicutyfedt'nF； Ara&fdcpsfs2,9? F4N2_9； FLORAL HOMEOTJC PROTEIN APETAL^lS^jmrnciPY Floral horn日otk gene gncDding a MADS domain protein hornolngous to SRF transcription Factors・ Specifies floral meristflrn and sopal identity, Required for the transcrptianal:a匚tivation of agiSMqus. [nter-acts with leafy Binds to promoter and regulate? the espressien of ftawenng time gen凹 SVP, £QIZ1 and AGL24.记住这个名称：AT1G69120这个就是APETALA1 (API)基因接下来开始查找APETALA1(AT1G69120)的突变体，拟南芥突变体库世界上有很多，公开的没有公开私用的都有，突变的方法也不 尽相同，有DS的，T-DNA插入的，Tosl7, EMS方法突变的等等。
但是，我们通常用美国 SALK 研究所的突变体库，这个突变体库比较权威，从这里可以找到几乎现有的所有拟南芥突变体，包括 T-DNA插入,RIKEN FST等等各种不同的突变类型，而且有详细的突变位点介绍和购买方法 它的搜索界面一目了然,使用也很方便下面介绍SALK突变体库的使用方法：Step 2：打开SALK主页:点击)，如下显示：Pld柑I J1丹肚TNI tn皿？JJumjff DN4 股血1km”SjJA: ^rabj'diwsij 1,001 处口 口 nK$SdlkHoiHigh ft曰口価ifot 此rjbbui吋旳g 屜佃皿Werre^cn斗 用右1鋼pj諭 :呵Rkc f'DMJCrjtHMf mmanii" J^labavr倉raMopsh T】也矚 ■Array TMKFrijrfume^raEHofa^m Suk ORFcxmw CtriJiKntibnKraitaiimEp讪Dunw Mmpy ArafrwJcpsTJ LiMLitT gcapjMif & Cikan.d 7ninirirrTp-rIim drirti w jeneiiLast tiodified ; Jsx 5. 2010 . t点击T-DNA Express进入(红圈处■y*mu-?ri町以调节图Fr^tiLdop&js 1-hsiJlsnEi CTRIF 冒刃■■ ■■^^™・・・・・ ™ ・・「 电」i：i -1■4- * T■吗CiLC.iii ^-7 曲・」rE»?*|j -rtl'—■ —H ■ — —■MrniEWttiBl t±S7££i tn 3 齐屮nJM>74iG丨.「..T. .. -..^*…FL履匚丫蛊-5- -!•S«LC_»»I1I7 W3.HIF-：*-—h. J一 <~ —!•凶爾#■"客仇卫」肚CW-l_：iKM> —!•SB_3_r!4Wm.E_2沖4 WL>_> h £ U jLHifdkixci^k i■ -aD- 3-li1flTgWMMM 壬冋，讣 SJSMl A>去i・」5的・r Mituwa* •van is-li-K-l_i 曲mi 1-431•L 札FaTiaL^KJ^-ajjruK.KLCC.Rk理5K/丄旳！riA3-K：MJ3+WlH-L7-mCM-|J»Wii.P#li4•> 12FATHlS-tWi-iJ H-M-TIIIZfflO.-Mj! 1*5hlTlLL-UfrJ-J JICb-k.fW^ZI fflftLKJS2Z7L屁T-*WL・w1PIL-«_F^ llFdOfi.«*+** !■_■«■ c«l_l Wlfa.jSeCETMH 121AT1G69120click here 临 他的不巻E-mdua"vbrta-6 *显示如下，所有信息全在如下窗口中从上述窗口中可以获得很多不同group制得的突变体，有SALK T-DNA, CSHL FST（冷泉港实验室的）等等，我个人建议使用SALK的突变体，订购比较方便，听同学说好像一百美元一个，上图中，蓝色下划线的那两个，以SALK —冠名的那个，两个显示的是不同的插入位置，和T-DNA插入方向（看在图中的位置和箭头方向）点击其中一个进入信息页，比如点击SALK_056708，得到如下页面:我们主要是从ABRC订购，点击进入页面，填写要求的相关信息，万事大吉。
显示的^T-DNA祝实验顺利！T-DNA Primer Design( Powered by GEBD )Please use the backup page served by AtTA, if the tdnaexpress server is down.The new T-DNA Primer Design Tool is now powered by Genome Express Browser Server (GEBD). The new tool can return the primers faster, and also give the insertion location information, the estimated T-DNA confirmation product size, as well as primer3-like format output. (July 28, 2005)Important Change: Now the RP is always on the side of the flanking sequence, that is, RP is always on the 3' end of the insertion. Therefore, the PCR reaction should always be set up as LB+RP for HM and LP+RP for WT. (Feb. 04, 2005)1. Protocol for SALK T-DNA primer designNote:N - Difference of the actual insertion site and the flanking sequence position, usually 0 - 300 bases MaxN - Maximum difference of the actual insertion site and the sequence, default 300 bps pZone - Regions used to pick up primers, default 100 bpsExt5, Ext3 - Regions between the MaxN to pZone, reserved not for picking up primers LP, RP - Left, Right genomic primerBP - T-DNA border primer LB - the left T-DNA border primer BPos - The distance from BP to the insertion siteLB - Left border primer of the T-DNA insertion:>LBbl of pBIN-pR0K2 for SALK linesGCGTGGACCGCTTGCTGCAACT> (Newly used by Salk Genotyping Project and with better resuIts) ATTTTGCCGATTTCGGAAC>LBa1 of pBIN-pROK2 for SALK linesTGGTTCACGTAGTGGGCCATCG >LB_6313R for SALK lines TCAAACAGGATTTTCGCCTGCT>LB1 for SAIL lines C/418-451 of pCSA110-pDAP101_T-DNAs GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC> >LB2 for SAIL lines C/390-423 of pCSA110-pDAP101_T-DNAs GCTTCCTATTATATCTTCCCAAATTACCAATACA>LB3 for SAIL lines C/350-383 of pCSA110-pDAP101_T-DNAs TAGCATCTGAATTTCATAACCAATCTCGATACACTo download SAIL pCSA110 & pDAP101 T-DNAs.By using the three primers +LP+RP) for SALK lines, users for WT (Wild Type - no insertion) should get a product of about 900-1100 bps ( from LP to RP ), for HM (Homozygous lines - insertions in both chromosomes) will get a band of 410+N bps ( from RP to insertion site 300+N bases, plus 110 bases from to the left border of the vector), and for HZ (Heterozygous lines - one of the pair chromosomes with insertion) will get both bands. The product size should be 200 base larger if using LBa1 instead of . However, the protocol requires thesame or similiar TM values for all the LB, LP and RP primers.You can set up two paired reactions, LP+RP and LB+RP. You should get a product in the LP+RP reaction for WT or HZ lines or get blank for HM lines, while get a band in the LB+RP for HM or HZ lines.。