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- 详细信息
- 技术资料
- 保存条件:
-20
- 保质期:
2年
- 英文名:
Plasmid#64608;NM_001278.3;IKBKAIKK-a
- 库存:
100
- 供应商:
上海烜雅生物科技有限公司
- 规格:
干粉/液体
基本信息
名称:IKKalpha (S176ES180E)-pcw107-V5人源基因质粒
别称: Plasmid#64608;NM_001278.3;IKBKAIKK-a
复制子: pUC
原核抗性: Amp
筛选标记:Puro
克隆菌株: Stbl3
培养条件: 37度
质粒属性
载体宿主:哺乳细胞
载体用途:蛋白表达
基因种属:人
基因类型:ORF
原核抗性:Amp
真核抗性:Puro
荧光蛋白:
质粒简介
KKalpha (S176E,S180E)-pcw107-V5人源基因质粒
when used to produce lentivirus, express physiological levels of insert
质粒图谱
质粒序列
LOCUS Exported 9880 bp ds-DNA circular SYN 20-JUL-2018
DEFINITION synthetic circular DNA
KEYWORDS IKKalpha (S176E,S180E)-pcw107-V5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9880)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9880
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..181
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 228..353
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 846..1079
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 1539..1749
/note="SV40 promoter"
/note="SV40 early promoter"
rep_origin 1608..1743
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 1789..2391
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAS"
misc_feature 2434..2551
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2609..3109
/note="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
protein_bind 3130..3154
/gene="mutant version of attB"
/bound_moiety="BP Clonase(TM)"
/note="attB1"
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 3172..5406
/note="IKKalpha (S176E,S180E)"
protein_bind complement(5408..5432)
/gene="mutant version of attB"
/bound_moiety="BP Clonase(TM)"
/note="attB2"
/note="recombination site for the Gateway(R) BP reaction"
CDS 5440..5481
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
misc_feature 5506..6094
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6166..6399
/note="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 6477..6598
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 6638..6773
/note="SV40 ori"
/note="SV40 origin of replication"
rep_origin 6980..7435
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7461..7565
/gene="bla"
/note="AmpR promoter"
CDS 7566..8426
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 8597..9185
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 9654..9880
/note="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
ORIGIN
1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccatgcattg catctcaatt
1501 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
1561 tgcgtctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
1621 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
1681 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
1741 gcctaggctt ttgcaaaaag ctttctagag gtaccaccgg agcttaccat gaccgagtac
1801 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
1861 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
1921 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
1981 tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag cgtcgaagcg
2041 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
2101 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
2161 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
2221 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
2281 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
2341 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccagtta acgaattcta
2401 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
2581 actttggcgc cggctcgagg cctgcaggtt gcgccttttc caaggcagcc ctgggtttgc
2641 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc
2701 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
2761 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
2821 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg
2881 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
2941 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
3001 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
3061 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg ggctagcatc
3121 gatggatcaa caagtttgta caaaaaagtt ggcccgggcg gcggcggcac catggagcgg
3181 cccccggggc tgcggccggg cgcgggcggg ccctgggaga tgcgggagcg gctgggcacc
3241 ggcggcttcg ggaacgtctg tctgtaccag catcgggaac ttgatctcaa aatagcaatt
3301 aagtcttgtc gcctagagct aagtaccaaa aacagagaac gatggtgcca tgaaatccag
3361 attatgaaga agttgaacca tgccaatgtt gtaaaggcct gtgatgttcc tgaagaattg
3421 aatattttga ttcatgatgt gcctcttcta gcaatggaat actgttctgg aggagatctc
3481 cgaaagctgc tcaacaaacc agaaaattgt tgtggactta aagaaagcca gatactttct
3541 ttactaagtg atatagggtc tgggattcga tatttgcatg aaaacaaaat tatacatcga
3601 gatctaaaac ctgaaaacat agttcttcag gatgttggtg gaaagataat acataaaata
3661 attgatctgg gatatgccaa agatgttgat caaggagaac tgtgtacaga atttgtggga
3721 acactgcagt atctggcccc agagctcttt gagaataagc cttacacagc cactgttgat
3781 tattggagct ttgggaccat ggtatttgaa tgtattgctg gatataggcc ttttttgcat
3841 catctgcagc catttacctg gcatgagaag attaagaaga aggatccaaa gtgtatattt
3901 gcatgtgaag agatgtcagg agaagttcgg tttagtagcc atttacctca accaaatagc
3961 ctttgtagtt taatagtaga acccatggaa aactggctac agttgatgtt gaattgggac
4021 cctcagcaga gaggaggacc tgttgacctt actttgaagc agccaagatg ttttgtatta
4081 atggatcaca ttttgaattt gaagatagta cacatcctaa atatgacttc tgcaaagata
4141 atttcttttc tgttaccacc tgatgaaagt cttcattcat tacagtctcg tattgagcgt
4201 gaaactggaa taaatactgg ttctcaagaa cttctttcag agacaggaat ttctctggat
4261 cctcggaaac cagcctctca atgtgttcta gatggagtta gaggctgtga tagctatatg
4321 gtttatttgt ttgataaaag taaaactgta tatgaagggc catttgcttc cagaagttta
4381 tctgattgtg taaattatat tgtacaggac agcaaaatac agcttccaat tatacagctg
4441 cgtaaagcgt gggctgaagc agtgcactat gtgtctggac taaaagaaga ctatagcagg
4501 ctctttcagg gacaaagggc agcaatgtta agtcttctta gatataatgc taacttaaca
4561 aaaatgaaga acactttgat ctcagcatca caacaactga aagctaaatt ggagtttttt
4621 cacaaaagca ttcagcttga cttggagaga tacagcgagc agatgacgta tgggatatct
4681 tcagaaaaaa tgctaaaagc atggaaagaa atggaagaaa aggccatcca ctatgctgag
4741 gttggtgtca ttggatacct ggaggatcag attatgtctt tgcatgctga aatcatggag
4801 ctacagaaga gcccctatgg aagacgtcag ggagacttga tggaatctct ggaacagcgt
4861 gccattgatc tatataagca gttaaaacac agaccttcag atcactccta cagtgacagc
4921 acagagatgg tgaaaatcat tgtgcacact gtgcagagtc aggaccgtgt gctcaaggag
4981 ctgtttggtc atttgagcaa gttgttgggc tgtaagcaga agattattga tctactccct
5041 aaggtggaag tggccctcag taatatcaaa gaagctgaca atactgtcat gttcatgcag
5101 ggaaaaaggc agaaagaaat atggcatctc cttaaaattg cctgtacaca gagttctgcc
5161 cggtcccttg taggatccag tctagaaggt gcagtaaccc ctcagacatc agcatggctg
5221 cccccgactt cagcagaaca tgatcattct ctgtcatgtg tggtaactcc tcaagatggg
5281 gagacttcag cacaaatgat agaagaaaat ttgaactgcc ttggccattt aagcactatt
5341 attcatgagg caaatgagga acagggcaat agtatgatga atcttgattg gagttggtta
5401 acagaattgc caactttctt gtacaaagtg gttaccggtg gtaagcctat ccctaaccct
5461 ctcctcggtc tcgattctac gtagtaatga ccgcgttaag tcgacaatca acctctggat
5521 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
5581 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
5641 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
5701 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
5761 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
5821 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
5881 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
5941 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
6001 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
6061 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
6121 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
6181 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
6241 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
6301 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
6361 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
6421 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
6481 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
6541 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
6601 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
6661 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
6721 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
6781 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
6841 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
6901 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
6961 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
7021 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
7081 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
7141 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
7201 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
7261 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
7321 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
7381 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
7441 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
7501 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
7561 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
7621 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
7681 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
7741 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
7801 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
7861 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
7921 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
7981 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
8041 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
8101 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
8161 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
8221 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
8281 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
8341 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
8401 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
8461 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
8521 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
8581 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
8641 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
8701 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
8761 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
8821 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
8881 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
8941 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
9001 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
9061 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
9121 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
9181 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
9241 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
9301 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
9361 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
9421 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
9481 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
9541 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
9601 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
9661 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
9721 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
9781 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
9841 cagagatatt gtatttaagt gcctagctcg atacataaac
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后,请先根据产品管壁标签来判断产品形式,并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉(常温运输,存于-20度,90天保质期,请务必转化挑单克隆培养,不要直接使用和测序)
①收到质粒干粉后请先5000rpm离心1min,再加入20μl ddH2O去离子水溶解质粒;
②取1支100μl 感受态于冰上解冻10min,加入2μl质粒,再冰浴30min后,42℃热激60s,不要搅动,再冰浴2min;(从第二步开始均要在超净工作台中无菌操作)
③加入900μl无抗的LB液体培养基,180rpm震荡37℃培养45min;
④6000rpm离心5min,仅留100μl上清液重悬细菌沉淀,并涂布至目标质粒抗性的LB平板上;(可使用本平台的平板涂布专用玻璃珠进行涂布,可以比传统涂布方法获得更多转化子)
⑤将平板正向培养1h,再倒置37℃培养14h。如果要求是30度则培养20h;
(菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态,要先转克隆感受态,重提质粒后再导入表达感受态)
⑥挑取单菌落至LB液体培养基中,加入对应抗生素,220rpm震荡培养14h,根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种(冰袋运输,存于-80℃,保质期90天,请务必划线挑单克隆培养)
四区划线后挑单菌落培养,酵母菌需要先液体复苏再四区划线,再挑单菌落液体培养。
3、穿刺菌种(冰袋运输,存于4℃,保质期7天)
穿刺接种,液体培养后四区划线,再挑单菌落液体培养。
4、菌落平板(冰袋运输,存于4℃,保质期7天)
直接挑取单菌落至液体培养基中。
5、液体质粒(冰袋运输,存于-20℃,保质期90天)
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒(常温运输,存于-20度,90天保质期,请务必转化挑单克隆培养,不要直接使用和测序)
收到货后将滤纸画圈部分剪下放入EP管中,加100ul无菌水将滤纸浸湿并浸泡5min,吸取5ul质粒转化,离心全涂。
二、转化图片
名称:IKKalpha (S176ES180E)-pcw107-V5人源基因质粒
别称: Plasmid#64608;NM_001278.3;IKBKAIKK-a
复制子: pUC
原核抗性: Amp
筛选标记:Puro
克隆菌株: Stbl3
培养条件: 37度
质粒属性
载体宿主:哺乳细胞
载体用途:蛋白表达
基因种属:人
基因类型:ORF
原核抗性:Amp
真核抗性:Puro
荧光蛋白:
质粒简介
KKalpha (S176E,S180E)-pcw107-V5人源基因质粒
when used to produce lentivirus, express physiological levels of insert
质粒图谱
质粒序列
LOCUS Exported 9880 bp ds-DNA circular SYN 20-JUL-2018
DEFINITION synthetic circular DNA
KEYWORDS IKKalpha (S176E,S180E)-pcw107-V5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9880)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9880
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..181
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 228..353
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 846..1079
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter 1539..1749
/note="SV40 promoter"
/note="SV40 early promoter"
rep_origin 1608..1743
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 1789..2391
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAS"
misc_feature 2434..2551
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2609..3109
/note="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
protein_bind 3130..3154
/gene="mutant version of attB"
/bound_moiety="BP Clonase(TM)"
/note="attB1"
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 3172..5406
/note="IKKalpha (S176E,S180E)"
protein_bind complement(5408..5432)
/gene="mutant version of attB"
/bound_moiety="BP Clonase(TM)"
/note="attB2"
/note="recombination site for the Gateway(R) BP reaction"
CDS 5440..5481
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
misc_feature 5506..6094
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6166..6399
/note="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 6477..6598
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 6638..6773
/note="SV40 ori"
/note="SV40 origin of replication"
rep_origin 6980..7435
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7461..7565
/gene="bla"
/note="AmpR promoter"
CDS 7566..8426
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 8597..9185
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 9654..9880
/note="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
ORIGIN
1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccatgcattg catctcaatt
1501 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
1561 tgcgtctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
1621 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
1681 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
1741 gcctaggctt ttgcaaaaag ctttctagag gtaccaccgg agcttaccat gaccgagtac
1801 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
1861 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
1921 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
1981 tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag cgtcgaagcg
2041 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
2101 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
2161 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
2221 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
2281 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
2341 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccagtta acgaattcta
2401 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
2581 actttggcgc cggctcgagg cctgcaggtt gcgccttttc caaggcagcc ctgggtttgc
2641 gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggtc
2701 tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg
2761 ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc
2821 gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg
2881 ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc
2941 agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt
3001 agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac
3061 gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg ggctagcatc
3121 gatggatcaa caagtttgta caaaaaagtt ggcccgggcg gcggcggcac catggagcgg
3181 cccccggggc tgcggccggg cgcgggcggg ccctgggaga tgcgggagcg gctgggcacc
3241 ggcggcttcg ggaacgtctg tctgtaccag catcgggaac ttgatctcaa aatagcaatt
3301 aagtcttgtc gcctagagct aagtaccaaa aacagagaac gatggtgcca tgaaatccag
3361 attatgaaga agttgaacca tgccaatgtt gtaaaggcct gtgatgttcc tgaagaattg
3421 aatattttga ttcatgatgt gcctcttcta gcaatggaat actgttctgg aggagatctc
3481 cgaaagctgc tcaacaaacc agaaaattgt tgtggactta aagaaagcca gatactttct
3541 ttactaagtg atatagggtc tgggattcga tatttgcatg aaaacaaaat tatacatcga
3601 gatctaaaac ctgaaaacat agttcttcag gatgttggtg gaaagataat acataaaata
3661 attgatctgg gatatgccaa agatgttgat caaggagaac tgtgtacaga atttgtggga
3721 acactgcagt atctggcccc agagctcttt gagaataagc cttacacagc cactgttgat
3781 tattggagct ttgggaccat ggtatttgaa tgtattgctg gatataggcc ttttttgcat
3841 catctgcagc catttacctg gcatgagaag attaagaaga aggatccaaa gtgtatattt
3901 gcatgtgaag agatgtcagg agaagttcgg tttagtagcc atttacctca accaaatagc
3961 ctttgtagtt taatagtaga acccatggaa aactggctac agttgatgtt gaattgggac
4021 cctcagcaga gaggaggacc tgttgacctt actttgaagc agccaagatg ttttgtatta
4081 atggatcaca ttttgaattt gaagatagta cacatcctaa atatgacttc tgcaaagata
4141 atttcttttc tgttaccacc tgatgaaagt cttcattcat tacagtctcg tattgagcgt
4201 gaaactggaa taaatactgg ttctcaagaa cttctttcag agacaggaat ttctctggat
4261 cctcggaaac cagcctctca atgtgttcta gatggagtta gaggctgtga tagctatatg
4321 gtttatttgt ttgataaaag taaaactgta tatgaagggc catttgcttc cagaagttta
4381 tctgattgtg taaattatat tgtacaggac agcaaaatac agcttccaat tatacagctg
4441 cgtaaagcgt gggctgaagc agtgcactat gtgtctggac taaaagaaga ctatagcagg
4501 ctctttcagg gacaaagggc agcaatgtta agtcttctta gatataatgc taacttaaca
4561 aaaatgaaga acactttgat ctcagcatca caacaactga aagctaaatt ggagtttttt
4621 cacaaaagca ttcagcttga cttggagaga tacagcgagc agatgacgta tgggatatct
4681 tcagaaaaaa tgctaaaagc atggaaagaa atggaagaaa aggccatcca ctatgctgag
4741 gttggtgtca ttggatacct ggaggatcag attatgtctt tgcatgctga aatcatggag
4801 ctacagaaga gcccctatgg aagacgtcag ggagacttga tggaatctct ggaacagcgt
4861 gccattgatc tatataagca gttaaaacac agaccttcag atcactccta cagtgacagc
4921 acagagatgg tgaaaatcat tgtgcacact gtgcagagtc aggaccgtgt gctcaaggag
4981 ctgtttggtc atttgagcaa gttgttgggc tgtaagcaga agattattga tctactccct
5041 aaggtggaag tggccctcag taatatcaaa gaagctgaca atactgtcat gttcatgcag
5101 ggaaaaaggc agaaagaaat atggcatctc cttaaaattg cctgtacaca gagttctgcc
5161 cggtcccttg taggatccag tctagaaggt gcagtaaccc ctcagacatc agcatggctg
5221 cccccgactt cagcagaaca tgatcattct ctgtcatgtg tggtaactcc tcaagatggg
5281 gagacttcag cacaaatgat agaagaaaat ttgaactgcc ttggccattt aagcactatt
5341 attcatgagg caaatgagga acagggcaat agtatgatga atcttgattg gagttggtta
5401 acagaattgc caactttctt gtacaaagtg gttaccggtg gtaagcctat ccctaaccct
5461 ctcctcggtc tcgattctac gtagtaatga ccgcgttaag tcgacaatca acctctggat
5521 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
5581 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
5641 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
5701 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
5761 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
5821 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
5881 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
5941 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
6001 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
6061 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
6121 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
6181 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
6241 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
6301 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
6361 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
6421 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
6481 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
6541 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
6601 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
6661 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
6721 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
6781 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
6841 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
6901 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
6961 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
7021 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
7081 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
7141 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
7201 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
7261 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
7321 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
7381 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
7441 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
7501 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
7561 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
7621 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
7681 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
7741 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
7801 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
7861 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
7921 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
7981 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
8041 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
8101 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
8161 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
8221 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
8281 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
8341 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
8401 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
8461 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
8521 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
8581 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
8641 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
8701 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
8761 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
8821 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
8881 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
8941 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
9001 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
9061 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
9121 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
9181 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
9241 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
9301 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
9361 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
9421 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
9481 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
9541 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
9601 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
9661 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
9721 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
9781 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
9841 cagagatatt gtatttaagt gcctagctcg atacataaac
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后,请先根据产品管壁标签来判断产品形式,并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉(常温运输,存于-20度,90天保质期,请务必转化挑单克隆培养,不要直接使用和测序)
①收到质粒干粉后请先5000rpm离心1min,再加入20μl ddH2O去离子水溶解质粒;
②取1支100μl 感受态于冰上解冻10min,加入2μl质粒,再冰浴30min后,42℃热激60s,不要搅动,再冰浴2min;(从第二步开始均要在超净工作台中无菌操作)
③加入900μl无抗的LB液体培养基,180rpm震荡37℃培养45min;
④6000rpm离心5min,仅留100μl上清液重悬细菌沉淀,并涂布至目标质粒抗性的LB平板上;(可使用本平台的平板涂布专用玻璃珠进行涂布,可以比传统涂布方法获得更多转化子)
⑤将平板正向培养1h,再倒置37℃培养14h。如果要求是30度则培养20h;
(菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态,要先转克隆感受态,重提质粒后再导入表达感受态)
⑥挑取单菌落至LB液体培养基中,加入对应抗生素,220rpm震荡培养14h,根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种(冰袋运输,存于-80℃,保质期90天,请务必划线挑单克隆培养)
四区划线后挑单菌落培养,酵母菌需要先液体复苏再四区划线,再挑单菌落液体培养。
3、穿刺菌种(冰袋运输,存于4℃,保质期7天)
穿刺接种,液体培养后四区划线,再挑单菌落液体培养。
4、菌落平板(冰袋运输,存于4℃,保质期7天)
直接挑取单菌落至液体培养基中。
5、液体质粒(冰袋运输,存于-20℃,保质期90天)
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒(常温运输,存于-20度,90天保质期,请务必转化挑单克隆培养,不要直接使用和测序)
收到货后将滤纸画圈部分剪下放入EP管中,加100ul无菌水将滤纸浸湿并浸泡5min,吸取5ul质粒转化,离心全涂。
二、转化图片
| P4123/pECMV-3×FLAG-RTN3人源基因质粒 |
| P4124/pECMV-3×FLAG-SCAMP5人源基因质粒 |
| P4125/pECMV-3×FLAG-SCAMP4人源基因质粒 |
| P4126/pECMV-3×FLAG-SLC26A1人源基因质粒 |
| P4127/pECMV-3×FLAG-SOD2人源基因质粒 |
| P4128/pECMV-3×FLAG-STX5人源基因质粒 |
| P4129/pECMV-3×FLAG-FAM3A人源基因质粒 |
| P4130/pECMV-3×FLAG-GLIPR1L1人源基因质粒 |
| P4131/pECMV-3×FLAG-DDIT4人源基因质粒 |
| P4132/pECMV-3×FLAG-ABHD4人源基因质粒 |
| P4133/pECMV-3×FLAG-CD69人源基因质粒 |
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IKKalpha (S176ES180E)-pcw107-V5人源基因质粒
¥100 - 1000










