拟南芥突变体购买流程-完全图解(共6页).doc
精选优质文档-倾情为你奉上最近要购买一批拟南芥突变体，想请教有经验的虫友购买拟南芥突变体的具体流程，例如我需要一个APETALA1的突变体，应到哪个网站进行搜索，怎样进行选择订购，越具体越好，有截图就更好了，谢谢大家了！l Step 1. 打开NCBI主页： 打开的页面如下：如下得到如下页面：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：记住这个名称：AT1G69120这个就是APETALA1（AP1）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，Tos17，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 T-DNA Express 进入(红圈处点击)，如下显示：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_，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。祝实验顺利！T-DNA Primer Design ( Powered by GEBD ) Please use 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 design Note:N - Difference of the actual insertion site and the flanking sequence position, usually 0 - 300 basesMaxN - Maximum difference of the actual insertion site and the sequence, default 300 bpspZone - 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 primerBPos - The distance from BP to the insertion site LB - Left border primer of the T-DNA insertion: > of pBIN-pROK2 for SALK lines GCGTGGACCGCTTGCTGCAACT> (Newly used by Salk Genotyping Project and with better results)ATTTTGCCGATTTCGGAAC> of pBIN-pROK2 for SALK lines TGGTTCACGTAGTGGGCCATCG>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 By using the three primers (LBb1.3+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 LBb1.3 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 LBb1.3. However, the protocol requires the same 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. 专心-专注-专业
