As with all other colors the preferred pattern has a white blaze that continues over the top of the head into the white area in the neck , a white end of the tail , white fore legs up till the elbow and white socks on the hind legs 所有颜色边境牧羊犬的首选斑纹样式都应该是? ?头部有一块白斑，从头顶延伸到脖子的白色区域，有白色的尾尖，白色的前腿（前腿上的白色向上至肘部）和好像穿着白袜子的后腿。
713bp and 700bp specific fragments were amplified by pcr and ligated into pgem - t easy vector . it was identified by restriction endonuclease digest analysis , pcr and sequencing that this fragment contained the complete open reading frame ( orf ) of the hc and ha gene 扩增产物连接到pgem - teasy载体上，转化入大肠杆菌jm109中进行蓝白斑筛选后，用酶切、 pcr鉴定和测序的方法鉴定出重组阳性质粒( pgem - hc和pgem - ha ) 。
John wyse nolan fell back with mr power , while martin cunningham took the elbow of a dapper little man in a shower of hail suit who walked uncertainly with hasty steps past micky anderson s watches . - the assistant town clerk s corns are giving him some trouble , john wyse nolan told mr power 约翰怀斯诺兰和鲍尔先生落在后面，马丁坎宁翰则挽住一位身穿带白斑点的深色衣服整洁而短小精悍的人，那个人正迈着急促的脚步趔趔趄趄地从米基安德森的钟表铺前走过。
Therefore , a - amylase has been used widely in many industrial fields , such as glucose production , beer brewing , fermentation trade , textile industry , and so on . the study on a - amylase is one of the most active fields in enzyme industrial fields . with the development of biotechnology , more and more scientists and researchers attempt to use dna shuffling technology to breed , screen and even " create " new a - amylase genes with higher activity and other new characters 设计引物时，上下游引物5 ’端分别添加了kpn和bamh酶切位点，克隆得到的基因片段和质粒载体都用kpn和bamh进行双酶切后，进行酶连、转化、筛选得到阳性重组子，经过蓝白斑验证、单酶切验证、双酶切验证、 pcr验证等一系列的验证后进行测序。
Through a series of experiments , the aging - resistance ability of hpch is increased one grade compared with the traditional materials , and the equal level with the foreign materials . the results of dry - wet circle test and quick test for chloride permeability show that the ability of efflorescence resistance of hpch is better . on the condition of 25 times circulation of dry - wet , there are a few non - development white spots just on the corner of specimens 通过干湿循环测试及快速氯离子渗透试验， hpch材料的抗析霜能力较强，在25次干湿循环条件下，仅在角部出现少量且不扩散的白斑，而传统装饰混凝土材料则出现延边棱迅速扩散的大面积析霜；快速氯离子渗透结果表明，在同样条件下hpch材料通过的电量(即离子迁移能力)仅为传统材料的49 . 68 ，比国外同期产品提高25 . 34 。
After electrophorised on 1 % agarose gel , the pcr production was purified with agarose gel dna extraction kit . the segment was ligated with vector pmd18 - t and then was tranformed into the competent cell of dh5 a . a construction mstnd - pmd18t was generated by inserting the sequence of 254bp into pmd18 - t vector and selecting the sense clones . positive clone was identified by three ways : endonuclease digestion , pcr and sequencing . the result showed that the cloned sequence coincides with the designed sequence . this construction was digested with nco i and xho i and ligated the pet28a ( + ) vector digested with the same enzymes using dna ligation kit . the production of ligation reaction was transformed into the competent cell of bl21 ( de3 ) . after 12 - 16 hours of culture , several colnes appeared on the plate . some positive clones were selected to extract their plasmid . these plasmids were digested by nco i and xho i and indentified by pcr . a contraction , mstnd - pet28a was generated . the result showed that the cloned sequence coincides with the designed sequence F _ 1长38bp ， r _ 1长36bp ，其它片段均40bp长， f _ 1和r _ 1片段两端分别加上限制性内切酶nco和xho的识别位点序列。用成对单链片段进行延伸反应，然后用其他单链片段作为引物，进行pcr扩增，用dna快速纯化回收试剂盒回收所得254bppcr产物，与pmd18 - t载体连接、转化dh _ 5 。受体菌感受态细胞，利用蓝白斑遗传学筛选法筛选阳性克隆，提取其质粒，采用nco和xho双酶切鉴定，获得了254bp的片段；用pmd18 - t载体上的特异引物rv - m和m13 - 47进行pcr鉴定，获得300bp的片段。
The 1 . 488kb dna fragment was sequenced and ligated to pbi121 vector and transformed into otsa deficient of e . coli strains ( ff4169 ) . otsa gene is in charge of trehalose - 6 - phosphate synthesis in e . coli . in growth curve experiment , the transformants that carried the 1 . 488kb dna fragment grew well in minimum medium , which contains 0 . 5mol / l nacl , while control strains could n ' t endure it 提取酿酒酵母的总dna ，以此为模板，采用pcr的方法从酿酒酵母中克隆出了1 . 488kb的海藻糖合成酶基因tps1片段，通过xba和sma双酶切，与同样经过xba和sma双酶切的puc118载体质粒连接，转入大肠杆菌dh5中，通过蓝白斑筛选重组子。
In order to construct the recombinant plasmid pcdna3 . 1 / ts87 , first , the ts87gene fragment was repcred using the redesigned primers to introduce re sites of hindld and bamh i , and kozak sequence . then the rebuilt ts87 fragment was cloned into t - vector and transformed into e . coli dh5 a 通过重新设计引物在ts87两端加上用于构建重组质粒的酶切位点hind和bamh和用于真核表达的kozak序列。将改造后的基因片段克隆入t - vector ，转化大肠杆菌dh5 ，通过蓝白斑筛选获得阳性克隆后测序鉴定。
Then , the plasmid was transformed into jm109 . the full length pstvd cdna recombinant plasmid was further identified by restriction mapping analysis and the nucleotide sequence of cdna cloned in pmd 18 - t vector was analyzed with ab1 377 dna sequence . test showed that the cdna of this chinese pstvd isolate was the same as pstvd - kf - 6 mild " type " isolate which originated from naturally infected potatoes in field of cornell university potato breeding program 利用rt - pgr技术，设计一对引物，对田间采集的经鉴定含pstvd的阳性样品进行全序列扩增，并将所得产物纯化回收，连接到pmd18t - vector中，并转化至大肠杆菌jm109中，挑选白斑进行双酶切( saci / ecorv )鉴定证明插入片段为359bp大小，进行序列测定，所得克隆基因为pstvd全序列负链，大小为359bp 。
The e2 gene was amplified by rt - pcr , then examined the fragment by electrophoresis . after purification and insertion into pucm - t vecter , the recombinant plasmid pbne2pi and pbne2pii were obtained . then they were transfected e . coli jm109 and screened positive clones by blue or white plaques . the recombinant plasmids were extracted and the inserted fragments were identificated by electrophoresis 通过rt - pcr扩增e _ 2基因，电泳测定扩增片段的大小，经纯化后，连接于pucm - t载体，获得重组质粒pbne2p 、 pbne2p ，转化e ? colijm109 ，经蓝白斑筛选，挑取阳性克隆，提取质粒，直接电泳鉴定和酶切鉴定。
