横向线性马达
Step 1. 打开NCBI主页：
打开的页⾯如下：
如下
旋转喷嘴得到如下页⾯：
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 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_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。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)scm文件
1. Protocol for SALK T-DNA primer design
动力换挡变速箱
Note:
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 bps
Ext5, Ext3 - Regions between the MaxN to pZone, reserved not for picking up primers
LP, RP - Left, Right genomic primer
BP - T-DNA border primer LB - the left T-DNA border primer
BPos - The distance from BP to the insertion site
LB - Left border primer of the T-DNA insertion:
乐器架>LBb1 of pBIN-pROK2 for SALK lines
GCGTGGACCGCTTGCTGCAACT
> (Newly used by Salk Genotyping Project and with better results)
ATTTTGCCGATTTCGGAAC
>LBa1 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
自动垃圾桶
>LB3 for SAIL lines C/350-383 of pCSA110-pDAP101_T-DNAs TAGCATCTGAATTTCATAACCAATCTCGATACAC
To 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 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.
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