供货周期: | 现货 |
品牌: | 泽叶生物 |
规格: | 2ug |
货号: | ZY6187 |
CAS号: |
pY004 (pcDNA3.1-hFnCpf1) 载体
质粒类型: | CRISPR/Cas系统 |
---|---|
启动子: | CMV |
载体大小: | 9443 bp (查看载体序列) |
载体抗性: | Ampicillin |
产品编号 | 产品名称 | 规格 | 价格 |
---|---|---|---|
ZY6187 | pY004 (pcDNA3.1-hFnCpf1) | 2ug | 点击询价 |
质粒图谱
载体序列:
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT
AAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA
ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCG
ATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC
ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC
ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA
TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC
AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA
CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTT
AAGCTTGGTACCGCCACCATGAGCATCTACCAGGAGTTCGTCAACAAGTATTCACTGAGTAAGACACTGC
GGTTCGAGCTGATCCCACAGGGCAAGACACTGGAGAACATCAAGGCCCGAGGCCTGATTCTGGACGATGA
GAAGCGGGCAAAAGACTATAAGAAAGCCAAGCAGATCATTGATAAATACCACCAGTTCTTTATCGAGGAA
ATTCTGAGCTCCGTGTGCATCAGTGAGGATCTGCTGCAGAATTACTCAGACGTGTACTTCAAGCTGAAGA
AGAGCGACGATGACAACCTGCAGAAGGACTTCAAGTCCGCCAAGGACACCATCAAGAAACAGATTAGCGA
GTACATCAAGGACTCCGAAAAGTTTAAAAATCTGTTCAACCAGAATCTGATCGATGCTAAGAAAGGCCAG
GAGTCCGACCTGATCCTGTGGCTGAAACAGTCTAAGGACAATGGGATTGAACTGTTCAAGGCTAACTCCG
ATATCACTGATATTGACGAGGCACTGGAAATCATCAAGAGCTTCAAGGGATGGACCACATACTTTAAAGG
CTTCCACGAGAACCGCAAGAACGTGTACTCCAGCAACGACATTCCTACCTCCATCATCTACCGAATCGTC
GATGACAATCTGCCAAAGTTCCTGGAGAACAAGGCCAAATATGAATCTCTGAAGGACAAAGCTCCCGAGG
CAATTAATTACGAACAGATCAAGAAAGATCTGGCTGAGGAACTGACATTCGATATCGACTATAAGACTAG
CGAGGTGAACCAGAGGGTCTTTTCCCTGGACGAGGTGTTTGAAATCGCCAATTTCAACAATTACCTGAAC
CAGTCCGGCATTACTAAATTCAATACCATCATTGGCGGGAAGTTTGTGAACGGGGAGAATACCAAGCGCA
AGGGAATTAACGAATACATCAATCTGTATAGCCAGCAGATCAACGACAAAACTCTGAAGAAATACAAGAT
GTCTGTGCTGTTCAAACAGATCCTGAGTGATACCGAGTCCAAGTCTTTTGTCATTGATAAACTGGAAGAT
GACTCAGACGTGGTCACTACCATGCAGAGCTTTTATGAGCAGATCGCCGCTTTCAAGACAGTGGAGGAAA
AATCTATTAAGGAAACTCTGAGTCTGCTGTTCGATGACCTGAAAGCCCAGAAGCTGGACCTGAGTAAGAT
CTACTTCAAAAACGATAAGAGTCTGACAGACCTGTCACAGCAGGTGTTTGATGACTATTCCGTGATTGGG
ACCGCCGTCCTGGAGTACATTACACAGCAGATCGCTCCAAAGAACCTGGATAATCCCTCTAAGAAAGAGC
AGGAACTGATCGCTAAGAAAACCGAGAAGGCAAAATATCTGAGTCTGGAAACAATTAAGCTGGCACTGGA
GGAGTTCAACAAGCACAGGGATATTGACAAACAGTGCCGCTTTGAGGAAATCCTGGCCAACTTCGCAGCC
ATCCCCATGATTTTTGATGAGATCGCCCAGAACAAAGACAATCTGGCTCAGATCAGTATTAAGTACCAGA
ACCAGGGCAAGAAAGACCTGCTGCAGGCTTCAGCAGAAGATGACGTGAAAGCCATCAAGGATCTGCTGGA
CCAGACCAACAATCTGCTGCACAAGCTGAAAATCTTCCATATTAGTCAGTCAGAGGATAAGGCTAATATC
CTGGATAAAGACGAACACTTCTACCTGGTGTTCGAGGAATGTTACTTCGAGCTGGCAAACATTGTCCCCC
TGTATAACAAGATTAGGAACTACATCACACAGAAGCCTTACTCTGACGAGAAGTTTAAACTGAACTTCGA
AAATAGTACCCTGGCCAACGGGTGGGATAAGAACAAGGAGCCTGACAACACAGCTATCCTGTTCATCAAG
GATGACAAGTACTATCTGGGAGTGATGAATAAGAAAAACAATAAGATCTTCGATGACAAAGCCATTAAGG
AGAACAAAGGGGAAGGATACAAGAAAATCGTGTATAAGCTGCTGCCCGGCGCAAATAAGATGCTGCCTAA
GGTGTTCTTCAGCGCCAAGAGTATCAAATTCTACAACCCATCCGAGGACATCCTGCGGATTAGAAATCAC
TCAACACATACTAAGAACGGGAGCCCCCAGAAGGGATATGAGAAATTTGAGTTCAACATCGAGGATTGCA
GGAAGTTTATTGACTTCTACAAGCAGAGCATCTCCAAACACCCTGAATGGAAGGATTTTGGCTTCCGGTT
TTCCGACACACAGAGATATAACTCTATCGACGAGTTCTACCGCGAGGTGGAAAATCAGGGGTATAAGCTG
ACTTTTGAGAACATTTCTGAAAGTTACATCGACAGCGTGGTCAATCAGGGAAAGCTGTACCTGTTCCAGA
TCTATAACAAAGATTTTTCAGCATACAGCAAGGGCAGACCAAACCTGCATACACTGTACTGGAAGGCCCT
GTTCGATGAGAGGAATCTGCAGGACGTGGTCTATAAACTGAACGGAGAGGCCGAACTGTTTTACCGGAAG
CAGTCTATTCCTAAGAAAATCACTCACCCAGCTAAGGAGGCCATCGCTAACAAGAACAAGGACAATCCTA
AGAAAGAGAGCGTGTTCGAATACGATCTGATTAAGGACAAGCGGTTCACCGAAGATAAGTTCTTTTTCCA
TTGTCCAATCACCATTAACTTCAAGTCAAGCGGCGCTAACAAGTTCAACGACGAGATCAATCTGCTGCTG
AAGGAAAAAGCAAACGATGTGCACATCCTGAGCATTGACCGAGGAGAGCGGCATCTGGCCTACTATACCC
TGGTGGATGGCAAAGGGAATATCATTAAGCAGGATACATTCAACATCATTGGCAATGACCGGATGAAAAC
CAACTACCACGATAAACTGGCTGCAATCGAGAAGGATAGAGACTCAGCTAGGAAGGACTGGAAGAAAATC
AACAACATTAAGGAGATGAAGGAAGGCTATCTGAGCCAGGTGGTCCATGAGATTGCAAAGCTGGTCATCG
AATACAATGCCATTGTGGTGTTCGAGGATCTGAACTTCGGCTTTAAGAGGGGGCGCTTTAAGGTGGAAAA
ACAGGTCTATCAGAAGCTGGAGAAAATGCTGATCGAAAAGCTGAATTACCTGGTGTTTAAAGATAACGAG
TTCGACAAGACCGGAGGCGTCCTGAGAGCCTACCAGCTGACAGCTCCCTTTGAAACTTTCAAGAAAATGG
GAAAACAGACAGGCATCATCTACTATGTGCCAGCCGGATTCACTTCCAAGATCTGCCCCGTGACCGGCTT
TGTCAACCAGCTGTACCCTAAATATGAGTCAGTGAGCAAGTCCCAGGAATTTTTCAGCAAGTTCGATAAG
ATCTGTTATAATCTGGACAAGGGGTACTTCGAGTTTTCCTTCGATTACAAGAACTTCGGCGACAAGGCCG
CTAAGGGGAAATGGACCATTGCCTCCTTCGGATCTCGCCTGATCAACTTTCGAAATTCCGATAAAAACCA
CAATTGGGACACTAGGGAGGTGTACCCAACCAAGGAGCTGGAAAAGCTGCTGAAAGACTACTCTATCGAG
TATGGACATGGCGAATGCATCAAGGCAGCCATCTGTGGCGAGAGTGATAAGAAATTTTTCGCCAAGCTGA
CCTCAGTGCTGAATACAATCCTGCAGATGCGGAACTCAAAGACCGGGACAGAACTGGACTATCTGATTAG
CCCCGTGGCTGATGTCAACGGAAACTTCTTCGACAGCAGACAGGCACCCAAAAATATGCCTCAGGATGCA
GACGCCAACGGGGCCTACCACATCGGGCTGAAGGGACTGATGCTGCTGGGCCGGATCAAGAACAATCAGG
AGGGGAAGAAGCTGAACCTGGTCATTAAGAACGAGGAATACTTCGAGTTTGTCCAGAATAGAAATAACAA
AAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCTACCCATACGATGTTCCA
GATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAGAAT
TCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC
GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGT
GCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA
TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA
TGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCC
TGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCC
TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT
AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG
GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGT
TCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT
ATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAAT
TAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCA
AAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATG
CAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC
CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGC
CTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTC
CCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAAC
AAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACA
GACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAG
ACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG
GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGT
GCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATG
CGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAG
CACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCC
AGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGAT
GCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTG
TGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC
TGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTT
GACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACG
AGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGG
ATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATA
ATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTG
TGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCG
TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCG
GAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT
GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC
GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC
GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA
CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG
CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT
AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGG
ATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGT
TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT
TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGG
TAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC
TACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA
GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG
AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA
CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA
GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC
ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG
ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG
ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTC
CATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT
GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC
AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT
CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT
GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA
TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAA
AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT
TCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAG
CAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACT
CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT
ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
一、实验原理:
碱裂解法提取质粒是根据共价闭合环状质粒DNA 与线性染色体DNA 在拓扑学上的差异来分离它们。在pH 值介于12.0 ~12.5 这个狭窄的范围内,线性的DNA 双螺旋结构解开而被变性,尽管在这样的条件下,共价闭环质粒DNA 的氢键会被断裂,但两条互补链彼此相互盘绕,仍会紧密地结合在一起。当加入pH 4.8 的乙酸钾高盐缓冲液恢复pH 至中性时,共价闭合环状的质粒DNA 的两条互补链仍保持在一起,因此复性迅速而准确,而线性的染色体DNA 的两条互补链彼此已完全分开,复性就不会那么迅速而准确,它们缠绕形成网状结构,通过离心,染色体DNA与不稳定的大分子RNA ,蛋白质-SDS 复合物等一起沉淀下来而被除去。
二、操作步骤:
1. 准备20ml灭菌过的培养管,编号,分别加入8ml LB培养基,再加入8μl 相应抗生素,可适量多加;
2. 用10μl 白色枪头挑取挑取单个菌落至培养管中,37℃震荡过夜(274rpm);
3. 取2ml过夜培养的菌液加入离心管中,室温10000rpm离心2min,去上清;(可重复步骤2,直至菌液离心完)(去上清时用纸吸一下)
4. 向留有菌体沉淀的离心管中加入250μl Buffer P1(含RNase A),使用移液枪充分混匀,悬浮菌体菌体;
5. 向离心管中加入250μl Buffer P2,温和地上下颠倒混匀4~6次,获得澄清的裂解液;(剧烈混合会使剪切染色体DNA,降低质粒纯度)
6. 向离心管中加入350μl Buffer N3, 立即温和地上下颠倒混匀4~6次,此时出现白色絮状沉淀。室温10000rpm离心15min;
7. 小心将步骤6中所得的上清液转移至洁净的吸收柱中。要保证没有吸入沉淀和细胞碎片。室温10000rpm离心1min,至裂解物完全通过吸收柱,倒掉收集管中的废液;
8. 向吸附柱中加150μl Buffer PB,10000rpm离心1min,倒掉收集管中的废液;
9. 向吸附柱中加400μl Buffer PW(检查是否加入无水乙醇),10000rpm离心1min,倒掉收集管中的废液。再空离一次,2min(此时可以加热 Buffer EB,65~70℃);
10. 将吸附柱置于一个新的离心管中,打开盖子,吹风4min左右(确保乙醇被去除,乙醇会影响下面的步骤);
11. 向吸附柱的吸附膜中间部位加90μl Buffer EB(pET系列拷贝数低,加EB 70μl即可),室温静置2min。10000rpm离心1min;
12. 把液体倒回吸附柱,在10000rpm离心1min;
13. 混合质粒至一个离心管中,做好标记,开盖室温放置两小时左右。-40℃保存质粒。
三、注意事项:
1.使用之前,把试剂盒中的一小管RNA酶加入Buffer P1, 4℃保存。
2.Buffer EB在65~70℃水浴预热,可以增加提取效率。
特别提示:本公司的所有产品仅可用于科研实验,严禁用于临床医疗及其他非科研用途!
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