擬南芥突變體購買流程-完全圖解

1、精選優(yōu)質(zhì)文檔-傾情為你奉上精選優(yōu)質(zhì)文檔-傾情為你奉上專心-專注-專業(yè)專心-專注-專業(yè)精選優(yōu)質(zhì)文檔-傾情為你奉上專心-專注-專業(yè)最近要購買一批擬南芥突變體，想請教有經(jīng)驗的蟲友購買擬南芥突變體的具體流程，例如我需要一個APETALA1的突變體，應(yīng)到哪個網(wǎng)站進行搜索，怎樣進行選擇訂購，越具體越好，有截圖就更好了，謝謝大家了！Step 1. 打開NCBI主頁： 打開的頁面如下：如下得到如下頁面：進一步獲得該基因在NCBI里面的基因信息，到此我為什么要做這一步呢，主要是想獲得該gene在擬南芥中的系統(tǒng)名，見下圖：記住這個名稱：AT1G69120這個就是APETALA1（AP1）基因接下來開始查找 APE

2、TALA1(AT1G69120)的突變體，擬南芥突變體庫世界上有很多，公開的沒有公開私用的都有，突變的方法也不盡相同，有DS的，T-DNA插入的，Tos17，EMS方法突變的等等。但是，我們通常用美國SALK研究所的突變體庫，這個突變體庫比較權(quán)威，從這里可以找到幾乎現(xiàn)有的所有擬南芥突變體，包括T-DNA插入,RIKEN FST等等各種不同的突變類型，而且有詳細的突變位點介紹和購買方法它的搜索界面一目了然,使用也很方便。下面介紹SALK突變體庫的使用方法：Step 2：打開SALK主頁：點擊 T-DNA Express 進入(紅圈處點擊)，如下顯示：顯示如下，所有信息全在如下窗口中從上述窗口中可

3、以獲得很多不同group制得的突變體，有SALK T-DNA，CSHL FST(冷泉港實驗室的)等等，我個人建議使用SALK的突變體，訂購比較方便，聽同學說好像一百美元一個，上圖中，藍色下劃線的那兩個，以SALK_冠名的那個，兩個顯示的是不同的插入位置，和T-DNA插入方向(看在圖中的位置和箭頭方向)點擊其中一個進入信息頁，比如點擊SALK_，得到如下頁面：我們主要是從 ABRC 訂購，點擊進入頁面，填寫要求的相關(guān)信息，萬事大吉。祝實驗順利！T-DNA Primer Design ( Powered by GEBD ) Please use served by AtTA, if the tdn

4、aexpress 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 2

5、8, 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

6、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 picki

7、ng 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

8、with better results)ATTTTGCCGATTTCGGAAC of pBIN-pROK2 for SALK lines TGGTTCACGTAGTGGGCCATCGLB_6313R for SALK lines TCAAACAGGATTTTCGCCTGCTLB1 for SAIL lines C/418-451 of pCSA110-pDAP101_T-DNAs GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC LB2 for SAIL lines C/390-423 of pCSA110-pDAP101_T-DNAs GCTTCCTATTATATCTTC

9、CCAAATTACCAATACALB3 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 line

10、s - 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 reac