| 出品公司: | ZYbscience |
|---|---|
| 载体名称: | pMD2.G |
| 质粒类型: | 哺乳动物载体；慢病毒包装载体；双质粒包装系统；信封载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 克隆方法: | 限制性内切酶，多克隆位点 |
| 启动子: | CMV |
| 载体大小: | 5824 BP |
| 5' 测序引物及序列: | CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’ |
| 3' 测序引物及序列: | -- |
| 载体标签: | 无 |
| 载体抗性: | Ampicillin |
| 筛选标记: | 无 |
| 克隆菌株: | DH5α或 HB101 |
| 宿主细胞（系）: | 包装细胞系如293T |
| 备注: | 慢病毒包装载体pMD2.G是2质粒包装系统的信封质粒与一起使用，适用范围、使用方法见下文。 也可以与第三代包装载体一同使用，构成3质粒包装系统。 |
| 产品目录号: | -- |
| 稳定性: | 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
产品详情
文献和实验
相关推荐
保存条件 ：-20℃低温保存
保质期 ：三年
英文名 ：pMD2.G
库存 ：20
供应商 ：泽叶生物
pMD2G载体基本信息
pMD2G载体质粒图谱和多克隆位点信息
pMD2G载体简介
第二代慢病毒包装质粒pMD2.G使用方法-慢病毒包装与转染方法Day 11. Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if desired)Day 21. Set up (use polypropylene tubes for this; polystyrene tubes DO NOT work!): 1 µg retroviral DNA encoding gene X 1 µg packaging plasmid Mix the packaging plasmid (psPAX2) at a 8:1 ratio with the envelope plasmid (pMD2.G)-a total of 1µg DME without serum to 94μL total 6 μL Fugene Mix and wait 15 to 30 minutes at room temperature Add to 293T cells without touching the sides of the dish (DO NOT CHANGE MEDIA) If you are using amphotropic virus then move immediately to BL2+ in a secondary container, which has an absorbent material. (This does not mean a couple of hours; it means Immediately!). The rest of this protocol is the same for all viruses---the BL2+ safety practices are in place if you are using amphotropic viruses Day 31. Change the media to whatever media you wish to use when infecting target cells. 293T cells are easily detached so remember not to put the media directly onto to cells, but rather “run” it down the side of the dish. Remember that you will get the highest titer virus when your cells are “happy.”2. Plate out your target cellsDay 41. Remove the medium from the 293T cells and use a 0.45 u syringe filter to remove any 293T cells. DO NOT use the 0.2 u filter, as it is likely to shear the envelope from your virusmaking it noninfectious Note: After filtering, the filter should be removed and placed in the biohazard bag in the hood and the syringe rinsed with bleach and decontaminated for a minimum of 20 min. It is useful to place a plastic beaker with bleach in the hood in advance2. Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target cells3. Carry out infection for 1 to 4 hours. Remove virus and replace with fresh media Note: If you wish to do a second infection the following day, it is important to put fresh media on the cells and not let the virus remain on the cells overnight. The media contains huge amounts of envelope, both associated and unassociated with viral particles, which will bind all the cell surface receptors required for virus adsorption, resulting in their down-regulation. Hence, if you don’t change the media after the initial infection, very few receptors will be available for the next round of infection. In addition, very few cells tolerate the presence of high levels of the VSV-G envelope for extended periods of time (i.e. a lot of your cells may die)Day 5 +1. Allow the cells to recover and begin to express the virus-encoded genes. The cells usually require 48 hours for this to occur2. Add drug if you are scoring for the presence of a vector that carries a drug resistance marker. Prior to this step it is advisable that you titrate the drug to be used for selection in order to know precisely how much to add. In addition, it is necessary to bring an extra plate of uninfected cells (often referred to as “canaries”) which will function as a positive control in the kill assay. Add drug to both plates. When your canaries are dead, you can remove the drug.
pMD2G载体序列
LOCUS pMD2.G 5824 bp DNA SYNDEFINITION pMD2.GACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTI http://www.biofeng.com/COMMENT VNTAUTHORNAME|biofeng.com|FEATURES Location/Qualifiers source 1..5824 /organism="pMD2.G" /mol_type="other DNA" misc_feature complement(40..59) /label="SV40pro_F_primer" promoter 162..738 /label="CMV_immearly_promoter" misc_feature 241..528 /label="CAG_enhancer" misc_feature 695..715 /label="CMV_fwd_primer" promoter 696..765 /label="CMV_promoter" promoter 708..827 /label="CMV2_promoter" misc_feature 739..758 /label="pCEP_fwd_primer" misc_feature 741..765 /label="LNCX_primer" misc_feature 1015..1398 /label="bGlob_int" gene 1450..2973 /label="vsv-G" /gene="vsv-G" CDS 1450..2985 /label="ORF frame 1" misc_feature 2950..2982 /label="VSVG Tag" rep_origin complement(4055..4674) /label="pBR322_origin" gene complement(4829..5689) /label="Ampicillin" /gene="Ampicillin" CDS complement(4829..5689) /label="ORF frame 1" promoter complement(5731..5759) /label="AmpR_promoter"ORIGIN 1 ggatcccctg agggggcccc catgggctag aggatccggc ctcggcctct gcataaataa 61 aaaaaattag tcagccatga gcttggccca ttgcatacgt tgtatccata tcataatatg 121 tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt 181 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 241 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 301 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 361 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 421 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 481 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 541 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 601 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 661 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 721 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 781 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctt 841 acatgtggta ccgagctcgg atcctgagaa cttcagggtg agtctatggg acccttgatg 901 ttttctttcc ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag 961 ggtacacata ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct 1021 tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc 1081 agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat 1141 aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg 1201 taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct 1261 tttattttat ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta 1321 atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg 1381 ctggcccatc actttggcaa agcacgtgag atctgaattc aacagagatc gatctgtttc 1441 cttgacacta tgaagtgcct tttgtactta gcctttttat tcattggggt gaattgcaag 1501 ttcaccatag tttttccaca caaccaaaaa ggaaactgga aaaatgttcc ttctaattac 1561 cattattgcc cgtcaagctc agatttaaat tggcataatg acttaatagg cacagccata 1621 caagtcaaaa tgcccaagag tcacaaggct attcaagcag acggttggat gtgtcatgct 1681 tccaaatggg tcactacttg tgatttccgc tggtatggac cgaagtatat aacacagtcc 1741 atccgatcct tcactccatc tgtagaacaa tgcaaggaaa gcattgaaca aacgaaacaa 1801 ggaacttggc tgaatccagg cttccctcct caaagttgtg gatatgcaac tgtgacggat 1861 gccgaagcag tgattgtcca ggtgactcct caccatgtgc tggttgatga atacacagga 1921 gaatgggttg attcacagtt catcaacgga aaatgcagca attacatatg ccccactgtc 1981 cataactcta caacctggca ttctgactat aaggtcaaag ggctatgtga ttctaacctc 2041 atttccatgg acatcacctt cttctcagag gacggagagc tatcatccct gggaaaggag 2101 ggcacagggt tcagaagtaa ctactttgct tatgaaactg gaggcaaggc ctgcaaaatg 2161 caatactgca agcattgggg agtcagactc ccatcaggtg tctggttcga gatggctgat 2221 aaggatctct ttgctgcagc cagattccct gaatgcccag aagggtcaag tatctctgct 2281 ccatctcaga cctcagtgga tgtaagtcta attcaggacg ttgagaggat cttggattat 2341 tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttccaatctc tccagtggat 2401 ctcagctatc ttgctcctaa aaacccagga accggtcctg ctttcaccat aatcaatggt 2461 accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca 2521 agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggca 2581 ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag 2641 tttcctttat acatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag 2701 gctcaggtgt tcgaacatcc tcacattcaa gacgctgctt cgcaacttcc tgatgatgag 2761 agtttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagcttgt agaaggttgg 2821 ttcagtagtt ggaaaagctc tattgcctct tttttcttta tcatagggtt aatcattgga 2881 ctattcttgg ttctccgagt tggtatccat ctttgcatta aattaaagca caccaagaaa 2941 agacagattt atacagacat agagatgaac cgacttggaa agtaactcaa atcctgcaca 3001 acagattctt catgtttgga ccaaatcaac ttgtgatacc atgctcaaag aggcctcaat 3061 tatatttgag tttttaattt ttatggaatt caccccacca gtgcaggctg cctatcagaa 3121 agtggtggct ggtgtggcta atgccctggc ccacaagttt cactaagctc gcttccttgc 3181 tgtccaattt ctattaaagg ttccttggtt ccctaagtcc aactactaaa ctgggggata 3241 ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 3301 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 3361 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 3421 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gccctgatgc 3481 ctatgcctta ttcatccctc agaaaaggat tcaagtagag gcttgatttg gaggttaaag 3541 tttggctatg ctgtatttta cattacttat tgttttagct gtcctcatga atgtcttttc 3601 actacccatt tgcttatcct gcatctctca gccttgactc cactcagttc tcttgcttag 3661 agataccacc tttcccctga agtgttcctt ccatgtttta cggcgagatg gtttctcctc 3721 gcctggccac tcagccttag ttgtctctgt tgtcttatag aggtctactt gaagaaggaa 3781 aaacaggggg catggtttga ctgtcctgtg agcccttctt ccctgcctcc cccactcaca 3841 gtgacccgga atccctcgac atggcagtct agcactagtg cggccgcaga tctgcttcct 3901 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3961 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 4021 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 4081 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4141 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4201 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4261 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4321 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4381 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4441 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4501 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4561 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4621 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4681 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4741 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4801 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4861 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4921 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4981 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 5041 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 5101 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 5161 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 5221 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 5281 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 5341 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 5401 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 5461 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 5521 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 5581 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 5641 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 5701 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5761 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5821 acgt
上海泽叶生物科技有限公司
实名认证
钻石会员
入驻年限：9年
