pcDNA3.1质粒图谱
pcDNA?3.1(+)
pcDNA?3.1(–)
Catalog nos. V790-20 and V795-20
Version K
10November2010
28-0104
User Manual
ii
Table of Contents
Important Information (v)
Accessory Products (vi)
Methods (1)
Overview (1)
Cloning into pcDNA?3.1 (2)
Transfection (6)
Creating Stable Cell Lines (7)
Appendix (10)
pcDNA?3.1 Vectors (10)
pcDNA?3.1/CAT (12)
Technical Support (13)
Purchaser Notification (13)
References (15)
iii
iv
Important Information
pcDNA?
Vectors
This manual is supplied with the following products.
Product Catalog no.
pcDNA?3.1(+) Vector V790-20
pcDNA?3.1(–) Vector V795-20
Shipping and Storage Vectors are shipped on wet ice. Upon receipt, store at –20°C.
Contents The pcDNA?3.1 vector components pcDNA?3.1 are listed below:
Item Concentration Volume
pcDNA?3.1 Vector pcDNA?3.1(+) or pcDNA?3.1(–)20 μg at 0.5 μg/μl, in TE buffer, pH 8.0
(10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
40 μl
Control Plasmid pcDNA?3.1/CAT 20 μg at 0.5 μg/μl, in TE buffer, pH 8.0
(10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
40 μl
Product Qualification The Certificate of Analysis provides detailed quality control information for each product. Certificates of Analysis are available on our website. Go to
https://www.wendangku.net/doc/7a17902423.html,/support and search for the Certificate of Analysis by product lot number, which is printed on the box.
v
Accessory Products
Additional Products Additional products that may be used with the pcDNA?3.1 vectors are available from Invitrogen. Ordering information is provided below.
Product Amount Catalog no.
One Shot? TOP10 Chemically
Competent Cells
10 reactions
20 reactions
C4040-10
C4040-03
One Shot? TOP10F’ Chemically
Competent Cells
20 reactions
40 reactions
C3030-03
C3030-06 Lipofectamine? 2000 1.5 ml
0.75 ml
11668-019
11668-027 Geneticin? 1 g
5 g
11811-023
11811-031 PureLink? HQ Mini Plasmid
Purification Kit
100 preps K2100-01
PureLink? HiPure Plasmid
Midiprep Kit
25 preps K2100-04
vi
Methods
Overview
Description pcDNA?3.1(+) and pcDNA?3.1(–) are 5.4 kb vectors derived from pcDNA?3 and designed for high-level stable and transient expression in mammalian hosts. High-
level stable and non-replicative transient expression can be carried out in most
mammalian cells. The vectors contain the following elements:
?Human cytomegalovirus immediate-early (CMV) promoter for high-level
expression in a wide range of mammalian cells
?Multiple cloning sites in the forward (+) and reverse (–) orientations to facilitate cloning
?Neomycin resistance gene for selection of stable cell lines
?Episomal replication in cells lines that are latently infected with SV40 or that
express the SV40 large T antigen (e.g. COS-1, COS-7)
The control plasmid, pcDNA?3.1/CAT, is included for use as a positive control for
transfection and expression in the cell line of choice.
Experimental Outline Use the following outline to clone and express your gene of interest in pcDNA?3.1. 1.Consult the multiple cloning sites described on pages 3-4 to design a strategy to
clone your gene into pcDNA?3.1.
2.Ligate your insert into the appropriate vector and transform into E. coli. Select
transformants on LB plates containing 50–100 μg/ml ampicillin.
3.Analyze your transformants for the presence of insert by restriction digestion.
4.Select a transformant with the correct restriction pattern and use sequencing to
confirm that your gene is cloned in the proper orientation.
5.Transfect your construct into the mammalian cell line of interest using your own
method of choice. Generate a stable cell line, if desired.
6.Test for expression of your recombinant gene by western blot analysis or
functional assay.
1
Cloning into pcDNA?3.1
Introduction Diagrams are provided on pages 3-4 to help you design a cloning strategy for
ligating your gene of interest into pcDNA?3.1. General considerations for cloning
and transformation are listed below.
General Molecular Biology Techniques For help with DNA ligations, E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, please refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).
E. coli Strain Many E. coli strains are suitable for the propagation of this vector including
TOP10F′, DH5 ?-T1R, and TOP10. We recommend that you propagate vectors
containing inserts in E. coli strains that are recombination deficient (rec A) and
endonuclease A-deficient (end A).
If you wish to express a human gene of interest from pcDNA?3.1, we recommend
using an Ultimate? Human ORF (hORF) Clone available from Invitrogen. For
more information about the Ultimate? hORF Clones available, refer to our Web
site (https://www.wendangku.net/doc/7a17902423.html,) or contact Technical Support(page 13). Transformation
Method
You may use any method of your choice for transformation. Chemical
transformation is the most convenient for most researchers. Electroporation is the
most efficient and the method of choice for large plasmids.
Maintenance of
pcDNA?3.1
To propagate and maintain pcDNA?3.1, use 10 ng of vector to transform a rec A,
end A E. coli strain like TOP10F′, DH5 ?-T1R, TOP10, or equivalent. Select
transformants on LB plates containing 50–100 μg/ml ampicillin. Be sure to
prepare a glycerol stock of your plasmid-containing E. coli strain for long-term
storage (see page 5).
Cloning
Considerations
pcDNA?3.1(+) and pcDNA?3.1(–) are non-fusion vectors. Your insert should
contain a Kozak translation initiation sequence with an ATG initiation codon for
proper initiation of translation (Kozak, 1987; Kozak, 1991; Kozak, 1990). An
example of a Kozak consensus sequence is provided below. Other sequences are
possible, but the G or A at position –3 and the G at position +4 (shown in bold)
illustrates the most commonly occurring sequence with strong consensus.
Replacing one of the two bases at these positions provides moderate consensus,
while having neither results in weak consensus. The ATG initiation codon is
shown underlined.
(G/A)NNATG G
Your insert must also contain a stop codon for proper termination of your gene.
Please note that the Xba I site contains an internal stop codon (TCTAGA).
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2
Multiple Cloning Site of
pcDNA?3.1(+) Below is the multiple cloning site for pcDNA?3.1(+). Restriction sites are labeled to indicate the cleavage site. The Xba I site contains an internal stop codon (TCTAGA). The multiple cloning site has been confirmed by sequencing and functional testing. The complete sequence of pcDNA? 3.1(+) is available for downloading from our web site (https://www.wendangku.net/doc/7a17902423.html,) or from Technical Support (see page 13). For a map and a description of the features of
pcDNA?3.1(+), please refer to the Appendix, pages 10-11.
1109
TCCTTTCCTA ATAAAATGAG GAAATTGCAT
Hin d
III
CAAT TA TA
T7 promoter/primer binding site
Bam H I Bst X I*
Eco R I Eco R V
Bst X I*
BGH poly (A) site
Nhe I Pme I
Apa I
Xho I
Not I
Kpn I
Afl II
Pme I
Xba I
Asp718I
pcDNA3.1/BGH reverse priming site CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG GACTCACTAT AGGGAGACCC AAGCTGGCTA GCGTTTAAAC TTAAGCTTGG TACCGAGCTC GGATCCACTA GTCCAGTGTG GTGGAATTCT GCAGATATCC AGCACAGTGG CGGCCGCTCG AGTCTAGAGG GCCCGTTTAA ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC
CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG
689
749
809
869
929
989
1049
enhancer region (3′ end)
*Please note that there are two Bst X I sites in the polylinker.
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3
4
Multiple Cloning
Site of
pcDNA ?3.1(–) Below is the multiple cloning site for pcDNA ?3.1(–). Restriction sites are labeled to indicate the cleavage site. The Xba I site contains an internal stop codon (TCTAGA). The multiple cloning site has been confirmed by sequencing and
functional testing. The complete sequence of pcDNA ?3.1(–) is available for
downloading from our web site (https://www.wendangku.net/doc/7a17902423.html, ) or from Technical
Support (see page 13). For a map and a description of the features of
pcDNA ?3.1(–), please see the Appendix , pages 10-11.
Hin d III CAAT TA TA T7 promoter/primer binding site Bam H I Bst X I*Eco R I Eco R V Bst X I*BGH poly (A) site Nhe I Pme I Apa I Xba I Xho I Not I Kpn I Afl II Pme I Asp 718 I pcDNA3.1/BGH reverse priming site CCTTTCCTAA TAAAATGAGG AAATTGCATC ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT GGTACCAAGC TTAAGTTTAA ACCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC GCCGCCACTG TGCTGGATAT CTGCAGAATT CCACCACACT GGACTAGTGG ATCCGAGCTC GACTCACTAT AGGGAGACCC AAGCTGGCTA GCGTTTAAAC GGGCCCTCTA GACTCGAGCG CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG 11091049
989
929
869
809
749
689
enhancer region (3′ end)
*Please note that there are two Bst X I sites in the polylinker.
E. coli Transformation Once you have obtained a clone containing your gene of interest, you may transform the clone into a suitable E. coli host (see below). We recommend including a negative control in your experiment to help you evaluate your results.
C We recommend that you sequence your construct with the T7 Promoter and BGH Reverse primers (Catalog nos. N560-02 and N575-02, respectively) to confirm that your gene is in the correct orientation for expression and contains an ATG and a stop codon. Please refer to the diagrams on pages 3-4 for the sequences and location of the priming sites. The primers are available separately from Invitrogen in 2 μg aliquots.
Preparing a Glycerol Stock Once you have identified the correct clone, purify the colony and make a glycerol stock for long-term storage. You should keep a DNA stock of your plasmid at –20°C.
?Streak the original colony out on an LB plate containing 50 μg/ml ampicillin.
Incubate the plate at 37°C overnight.
?Isolate a single colony and inoculate into 1–2 ml of LB containing 50 μg/ml ampicillin.
?Grow the culture to mid-log phase (OD600 = 0.5–0.7).
?Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.
Store at –80°C.
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5
Transfection
Introduction Once you have verified that your gene is cloned in the correct orientation and contains an initiation ATG and a stop codon, you are ready to transfect your cell
line of choice. We recommend that you include the positive control vector and a
mock transfection (negative control) to evaluate your results.
Plasmid Preparation Once you have generated your expression clone, you must isolate plasmid DNA for transfection. Plasmid DNA for transfection into eukaryotic cells must be clean and free contamination with from phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using the PureLink? HQ Mini Plasmid Purification Kit (Catalog no. K2100-01), the PureLink? HiPure Plasmid Midiprep Kit (Catalog no. K2100-04), or CsCl gradient centrifugation.
Methods of Transfection For established cell lines (e.g. HeLa), consult original references or the supplier of your cell line for the optimal method of transfection. We recommend that you follow exactly the protocol for your cell line. Pay particular attention to medium requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology (Ausubel et al., 1994).
Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). For high efficiency transfection in a broad range of mammalian cell lines, we recommend using Lipofectamine? 2000 Reagent (Catalog no. 11668-027) available from Invitrogen. For more information about Lipofectamine? 2000 and other transfection reagents, refer to our Web site (https://www.wendangku.net/doc/7a17902423.html,) or contact Technical Support (page 13).
Positive Control pcDNA?3.1/CAT is provided as a positive control vector for mammalian
transfection and expression (see page 12) and may be used to optimize transfection
conditions for your cell line. The gene encoding chloramphenicol acetyl transferase
(CAT) is expressed in mammalian cells under the control of the CMV promoter. A
successful transfection will result in CAT expression that can be easily assayed (see
below).
Assay for CAT Protein You may assay for CAT expression by ELISA assay, western blot analysis, fluorometric assay, or radioactive assay (Ausubel et al., 1994; Neumann et al., 1987). If you wish to detect CAT protein using western blot analysis, you may use the Anti-CAT Antiserum (Catalog no. R902-25) available from Invitrogen. Other kits to assay for CAT protein using ELISA assay are available from Roche Molecular Biochemicals (Catalog no. 1 363 727) and Molecular Probes (Catalog no. F-2900).
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6
Creating Stable Cell Lines
Introduction The pcDNA?3.1(+) and pcDNA?3.1(–) vectors contain the neomycin resistance gene for selection of stable cell lines using neomycin (Geneticin?). We recommend that you
test the sensitivity of your mammalian host cell to Geneticin? as natural resistance
varies among cell lines. General information and guidelines are provided in this
section for your convenience.
C To obtain stable transfectants, we recommend that you linearize your pcDNA?3.1 construct before transfection. While linearizing the vector may not improve the efficiency of transfection, it increases the chances that the vector does not integrate in a way that disrupts elements necessary for expression in mammalian cells. To linearize your construct, cut at a unique site that is not located within a critical element or within your gene of interest.
Geneticin?Geneticin? blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an aminoglycoside, similar in structure to neomycin, gentamycin, and
kanamycin. Expression in mammalian cells of the bacterial aminoglycoside
phosphotransferase gene (APH), derived from Tn5, results in detoxification of
Geneticin? (Southern and Berg, 1982).
Determining Antibiotic Sensitivity To successfully generate a stable cell line expressing your protein of interest, you need to determine the minimum concentration of Geneticin? required to kill your untransfected host cell line. Test a range of concentrations (see protocol below) to ensure that you determine the minimum concentration necessary for your cell line. 1. Plate or split a confluent plate so the cells will be approximately 25% confluent. Prepare a set
of 6–7 plates. Add the following concentrations of antibiotic to each plate:
?For Geneticin? selection, test 0, 50, 125, 250, 500, 750, and 1000 μg/ml Geneticin?.
2. Replenish the selective media every 3–4 days, and observe the percentage of surviving cells.
3. Count the number of viable cells at regular intervals to determine the appropriate
concentration of antibiotic that prevents growth within 1–3 weeks after addition of the
antibiotic.
Geneticin?Selection Guidelines Once you have determined the appropriate Geneticin? concentration to use for selection, you can generate a stable cell line expressing your pcDNA?3.1 construct. Geneticin? is available separately from Invitrogen (see page vi for ordering information). Use as follows:
1.Prepare Geneticin? in a buffered solution (e.g. 100 mM HEPES, pH 7.3).
https://www.wendangku.net/doc/7a17902423.html,e the predetermined concentration of Geneticin? in complete medium.
3.Calculate concentration based on the amount of active drug.
4.Cells will divide once or twice in the presence of lethal doses of Geneticin?, so the effects of
the drug take several days to become apparent. Complete selection can take from 2 to 3
weeks of growth in selective medium.
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8
Possible Sites for Linearization of pcDNA3.1(+) Prior to transfection, we recommend that you linearize the pcDNA ?3.1(+) vector. Linearizing pcDNA ?3.1(+) will decrease the likelihood of the vector integrating into the genome in a way that disrupts the gene of interest or other elements required for expression
in mammalian cells. The table below lists unique restriction sites that may be used to
linearize your construct prior to transfection. Other unique restriction sites are possible. Be
sure that your insert does not contain the restriction enzyme site you wish to use to linearize
your vector.
Enzyme R estriction Site
(bp)
Location Supplier Bgl II
12 Upstream of CMV promoter Invitrogen, Catalog no. 15213-028 Mfe I
161 Upstream of CMV promoter New England Biolabs Bst 1107 I
3236 End of SV40 polyA AGS *, Fermentas, Takara, Roche Mol. Biochemicals Eam 1105 I
4505 Ampicillin gene AGS *, Fermentas, Takara Pvu I
4875 Ampicillin gene Invitrogen, Catalog no. 25420-019 Sca I
4985 Ampicillin gene Invitrogen, Catalog no. 15436-017 Ssp I 5309 bla promoter
Invitrogen, Catalog no. 15458-011
Possible Sites for Linearization of pcDNA ?3.1(–) The table below lists unique restriction sites that may be used to linearize your pcDNA ?3.1(–) construct prior to transfection. Other unique restriction sites are possible. Be sure that your insert does not contain the restriction enzyme site you wish to use to
linearize your vector.
Enzyme R estriction Site
(bp)
Location Supplier Bgl II
12 Upstream of CMV promoter Invitrogen, Catalog no. 15213-028 Mfe I
161 Upstream of CMV promoter New England Biolabs Bst 1107 I
3235 End of SV40 polyA AGS *, Fermentas, Takara, Roche Mol. Biochemicals Eam 1105 I
4504 Ampicillin gene AGS *, Fermentas, Takara Pvu I
4874 Ampicillin gene Invitrogen, Catalog no. 25420-019 Sca I
4984 Ampicillin gene Invitrogen, Catalog no. 15436-017 Ssp I 5308 bla promoter
Invitrogen, Catalog no. 15458-011 *Angewandte Gentechnologie Systeme
continued on next page
Selection of Stable Integrants Once you have determined the appropriate Geneticin? concentration to use for selection in your host cell line, you can generate a stable cell line expressing your gene of interest.
1.Transfect your mammalian host cell line with your pcDNA?3.1 construct using the desired
protocol. Remember to include a plate of untransfected cells as a negative control and the pcDNA?3.1/CAT plasmid as a positive control.
2.24 hours after transfection, wash the cells and add fresh medium to the cells.
3.48 hours after transfection, split the cells into fresh medium containing Geneticin? at the pre-
determined concentration required for your cell line. Split the cells such that they are no more than 25% confluent.
4.Feed the cells with selective medium every 3–4 days until Geneticin?-resistant foci can be
identified.
5.Pick and expand colonies in 96- or 48-well plates.
9
10 Appendix
pcDNA?3.1 Vectors Map The figure below summarizes the features of the pcDNA ?3.1(+) and pcDNA ?3.1(–)
vectors. The complete sequences for pcDNA ?3.1(+) and pcDNA ?3.1(–) are available
for down-loading from our World Wide Web site (https://www.wendangku.net/doc/7a17902423.html, ) or from
Technical Support (see page 13). Details of the multiple cloning sites are shown on
page 3 for pcDNA ?3.1(+) and page 4 for pcDNA ?3.1(–).
Comments for pcDNA3.1 (+) 5428 nucleotides
CMV promoter: bases 232-819T7 promoter/priming site: bases 863-882Multiple cloning site: bases 895-1010
BGH polyadenylation sequence: bases 1028-1252f1 origin: bases 1298-1726SV40 early promoter and origin: bases 1731-2074Neomycin resistance gene (ORF): bases 2136-2930SV40 early polyadenylation signal: bases 3104-3234pUC origin: bases 3617-4287 (complementary strand)Ampicillin resistance gene (bla ): bases 4432-5428 (complementary strand) ORF: bases 4432-5292 (complementary strand) Ribosome binding site: bases 5300-5304 (complementary strand) bla promoter (P3): bases 5327-5333 (complementary strand) I I (+)
( ) continued on next page
pcDNA?3.1 Vectors, continued
Features pcDNA?3.1(+) (5428 bp) and pcDNA?3.1(–) (5427 bp) contain the following elements.
All features have been functionally tested.
Feature Benefit
Human cytomegalovirus (CMV) immediate-early promoter/ enhancer Permits efficient, high-level expression of your recombinant protein (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987)
T7 promoter/priming site Allows for in vitro transcription in the sense
orientation and sequencing through the insert
Multiple cloning site in forward or reverse orientation Allows insertion of your gene and facilitates cloning
Bovine growth hormone (BGH) polyadenylation signal Efficient transcription termination and polyadenylation of mRNA (Goodwin and Rottman, 1992)
f1 origin Allows rescue of single-stranded DNA
SV40 early promoter and origin Allows efficient, high-level expression of the
neomycin resistance gene and episomal replication
in cells expressing SV40 large T antigen Neomycin resistance gene Selection of stable transfectants in mammalian
cells (Southern and Berg, 1982)
SV40 early polyadenylation signal Efficient transcription termination and
polyadenylation of mRNA
pUC origin High-copy number replication and growth in
E. coli
Ampicillin resistance gene
(β-lactamase)
Selection of vector in E. coli
Ampicillin (bla) resistance gene
(β-lactamase)
Allows selection of transformants in E. coli
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pcDNA?3.1/CAT
Description pcDNA?3.1/CAT is a 6217 bp control vector containing the gene for CAT. It was constructed by digesting pcDNA?3.1(+) with Xho I and Xba I and treating with
Klenow. An 800 bp Hin d III fragment containing the CAT gene was treated with
Klenow and then ligated into pcDNA?3.1(+).
Map The figure below summarizes the features of the pcDNA?3.1/CAT vector. The complete nucleotide sequence for pcDNA?3.1/CAT is available for downloading
from our World Wide Web site (https://www.wendangku.net/doc/7a17902423.html,) or by contacting Technical
Support (see page 13).
Comments for pcDNA3.1(+)/CAT
6217 nucleotides
CMV promoter: bases 232-819
CAT ORF: bases 1027-1686
pcDNA3.1/BGH reverse priming site: bases 1811-1828
BGH polyadenylation sequence: bases 1817-2041
f1 origin: bases 2087-2515
SV40 early promoter and origin: bases 2520-2863
Neomycin resistance gene (ORF): bases 2925-3719
SV40 early polyadenylation sequence: bases 3893-4023
pUC origin: bases 4406-5076 (complementary strand)
Ampicillin resistance gene (ORF): bases 5221-6081 (complementary strand)
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Technical Support
World Wide
Visit the Invitrogen website at https://www.wendangku.net/doc/7a17902423.html, for:
?Technical resources, including manuals, vector maps and sequences, application notes, MSDSs, FAQs, formulations, citations, handbooks, etc.
?Complete technical support contact information
?Access to the Invitrogen Online Catalog
Additional product information and special offers
Contact Us For more information or technical assistance, call, write, fax, or email. Additional international offices are listed on our Web page (https://www.wendangku.net/doc/7a17902423.html,).
Corporate Headquarters:Japanese Headquarters: European Headquarters:
Invitrogen Corporation Invitrogen Japan K.K. Invitrogen Ltd
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Fax: 1 760 602 6500Fax: 81 3 3663 8242 Tech Fax: +44 (0) 141 814 6117
E-mail: tech_support@https://www.wendangku.net/doc/7a17902423.html, E-mail: jpinfo@https://www.wendangku.net/doc/7a17902423.html, E-mail: eurotech@https://www.wendangku.net/doc/7a17902423.html,
MSDS Material Safety Data Sheets (MSDSs) are available on our website at
https://www.wendangku.net/doc/7a17902423.html,/msds.
Certificate of Analysis The Certificate of Analysis (CofA) provides detailed quality control information for each product. CofAs are available on our website at https://www.wendangku.net/doc/7a17902423.html,/support, and are searchable by product lot number, which is printed on each box.
Limited Warranty Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, contact our Technical Support Representatives. Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Corporation’s liability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Support Representatives.
Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.
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Purchaser Notification
13
Introduction Use of pcDNA?3.1 is covered under the licenses detailed below.
Limited Use Label License No. 5: Invitrogen Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer(whether the buyer is an academic or for-profit entity).The buyer cannot sell or otherwise transfer(a)this product(b)its components or(c)materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes.The buyer may transfer information or materials made through the use of this product to a scientific collaborator,provided that such transfer is not for any Commercial Purpose,and that such collaborator agrees in writing(a)not to transfer such materials to any third party,and(b)to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include,but is not limited to:(1)use of the product or its components in manufacturing;(2)use of the product or its components to provide a service,information,or data;(3)use of the product or its components for therapeutic,diagnostic or prophylactic purposes;or(4)resale of the product or its components,whether or not such product or its components are resold for use in research.For products that are subject to multiple limited use label licenses,the terms of the most restrictive limited use label license shall control.Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture,use or sale of a therapeutic,clinical diagnostic,vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed,provided that neither this product nor any of its components was used in the manufacture of such product.If the purchaser is not willing to accept the limitations of this limited use statement,Life Technologies is willing to accept return of the product with a full refund.For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing,Life Technologies,5791Van Allen Way,Carlsbad,California92008;Phone(760)603-7200or e-mail:outlicensing@https://www.wendangku.net/doc/7a17902423.html,.
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如何阅读分析质粒图谱
如何阅读分析质粒图谱 日期：2012-04-18来源：未知作者：xilu点击：次 如何阅读分析质粒图谱 载体主要有病毒和非病毒两大类,其中质粒DNA是一种新的非病毒转基因载体。 一个合格质粒的组成要素 1. 复制起始位点Ori即控制复制起始的位点。原核生物DNA分子中只有一个复制起始点。而真核生物DNA分子有多个复制起始位点。 2. 抗生素抗性基因可以便于加以检测，如Amp+ ,Kan+ 3. 多克隆位点MCS 克隆携带外源基因片段 4. P/E 启动子/增强子 5. Terms 终止信号 6. 加poly(A)信号可以起到稳定mRNA作用 如何阅读质粒图谱 第一步：首先看Ori的位置，了解质粒的类型(原核/真核/穿梭质粒) 所谓穿梭质粒是指一类人工构建的具有两种不同复制起点和选择标记，因而可以在两种不同类群宿主中存活和复制的质粒载体。此概念不仅用于不同的微生物菌群之间，也可以推广到真核生物表达载体的构建，如用于枯草的pBE2、酵母的pPIC9K、哺乳动物表达载体pMT2 和用于植物细胞的Ti 质粒。这些穿梭质粒不仅可以在大肠杆菌中复制扩增，也可以在相应的枯草、酵母、动物或植物细胞中扩增和表达。这样利于对质粒的分子生物学操作和大量制备。 第二步：再看筛选标记，如抗性，决定使用什么筛选标记。 1. Ampr水解β-内酰胺环，解除氨苄的毒性。 2. tetr可以阻止四环素进入细胞。 3. camr生成氯霉素羟乙酰基衍生物，使之失去毒性。
4. neor(kanr) 氨基糖苷磷酸转移酶使G418(长那霉素衍生物)失活 5. hygr使潮霉素β失活。 第三步：看多克隆位点(MCS)。它具有多个限制酶的单一切点。便于外源基因的插入。如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，而便于筛选。决定能不能放目的基因以及如何放置目的基因。 第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外源DNA片段。一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。 第五步：是否含有表达系统元件，即启动子-核糖体结合位点-克隆位点-转录终止信号。这是用来区别克隆载体与表达载体。克隆载体中加入一些与表达调控有关的元件即成为表达载体。选用那种载体，还是要以实验目的为准绳。 启动子-核糖体结合位点-克隆位点-转录终止信号
质粒图谱信息
质粒图谱信息 一.九种表达载体 Pllp-OmpA, pllp-STII, pMBP-P, pMBP-C, pET-GST, pET-Trx, pET-His, pET-CKS, pET-DsbA 二.克隆载体 pTZ19R DNA pUC57 DNA PMD18T PQE30 pUC18 pUC19 pTrcHisA pTrxFus pRSET-A pRSET-B pV AX1 PBR322 pbv220 pBluescript II KS (+) L4440 pCAMBIA-1301 pMAL-p2X pGD926 三.PET系列表达载体 Protein Expression ? Prokaryotic Expression ? pETDsb Fusion Systems 39b and 40b Protein Expression ? Prokaryotic Expression ? pET Expression System 33b Protein Expression ? Prokaryotic Expression ? pET Expression Systems Protein Expression ? Prokaryotic Expression ? pET Expression Systems plus Competent Cells Protein Expression ? Prokaryotic Expression ? pET GST Fusion Systems 41 and 42 Protein Expression ? Prokaryotic Expression ? pETNusA Fusion Systems 43.1 and 44 Protein Expression ? Prokaryotic Expression ? pET Vector DNA Protein Purification ? Purification Systems ? Strep?Tactin Resins and Purification Kits 四.PGEX系列表达载体 T EcoR pGEX-1 I/BAP pGEX-2T pGEX-2TK pGEX-3X pGEX-4T-1 pGEX-4T-2 pGEX-4T-3 pGEX-5X-1 pGEX-5X-2 pGEX-5X-3
维真生物-如何阅读基因载体图谱
如何阅读基因载体图谱 基因载体是基因工程的核心，也是基因治疗中强有力的生物工具，我们先来认识和阅读载体图谱吧。 一、载体分类及载体组成元件 载体分类 1、按属性分类：病毒载体和非病毒载体 病毒载体是一种常见的分子生物学工具，可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入目的细胞，进行感染的分子机制。可发生于完整活体或是细胞培养中。可应用于基础研究、基因疗法或疫苗。用于基因治疗和疫苗的病毒载体应具备以下基本条件： （1）携带外源基因并能包装成病毒颗粒； （2）介导外源基因的转移和表达； （3）对人体不致病； （4）在环境中不会引起增殖和传播。 非病毒载体一般是指质粒DNA。 2、按进入受体细胞的类型分类：原核载体、真核载体、穿梭载体（含原核和真核2个复制子，能在原核和真核细胞中复制，并可以在真核细胞中有效表达）。 3、按功能分类：克隆载体、表达载体 克隆载体：具有克隆载体的基本元件（Ori，Ampr，MCS等），可以携带DNA片段或外源基因进入受体细胞并克隆和大量扩增DNA片段（外源基因）的载体。 表达载体：克隆载体中加入一些与表达调控（具有转录/翻译所必需的DNA顺序）有关的元件即成为表达载体。 载体组成元件 1、复制起始位点Ori：即控制复制起始的位点。Ori的箭头指复制方向，其他元件标注的箭头多指转录方向（正向）。 2、抗生素抗性基因：可以便于加以检测，如Amp+ ，Kan+ （1）Ampr：水解β-内酰胺环，解除氨苄的毒性。
（2）tetr ：可以阻止四环素进入细胞。 （3）camr：生成氯霉素羟乙酰基衍生物，使之失去毒性。 （4）neor（kanr）：氨基糖苷磷酸转移酶，使G418（卡那霉素衍生物）失活。 （5）hygr：使潮霉素β失活。 3、多克隆位点：MCS克隆携带外源基因片段，它具有多个限制酶的单一切点，便于外源基因的插入。如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，便于筛选。决定能不能放目的基因以及如何放置目的基因。还要再看外源DNA插入片段大小。质粒一般只能容纳小于10kb的外源DNA片段。一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。 4、P/E：启动子/增强子 5、Terms：终止信号 6、加poly（A）信号：可以起到稳定mRNA作用 示例阅读载体： pENTER载体 1）human ORF + pENTER载体 2） CMV启动子，T7启动子 3） ORF的C端融合了Flag和His tag 4) 多克隆位点，常用AsisI 和 MluI(人源基因上不常见的)
如何阅读质粒图谱(更新版本)
如何阅读质粒图谱 最近由于实验需要,需要查阅载体图谱,到园子里搜罗一番,发现虽然有人问载体图谱阅读的问题,也有前辈回答,但都不详细,借自己也在琢磨这个问题的机会,将我学到的东西整理一下,于 大家分享。 载体主要有病毒和非病毒两大类,其中质粒DNA是一种新的非病毒转基因载体。 一个合格质粒的组成要素 #复制起始位点Oril 即控制复制起始的位点。原核生物DNA分子中只有一个复制起始点。而真核生物DNA分子有多个复制起始位点。 ＃抗生素抗性基因可以便于加以检测，如Amp+l ,Kan+ ＃多克隆位点MCS 克隆携带外源基因片段l ＃P/E 启动子/增强子l ＃Termsl 终止信号 ＃加poly（A）信号l 可以起到稳定mRNA作用 二、如何阅读质粒图谱 第一步：首先看Ori的位置，了解质粒的类型（原核/真核/穿梭质粒） 第二步：再看筛选标记，如抗性，决定使用什么筛选标记。 （1）Ampr 水解β－内酰胺环，解除氨苄的毒性。 （2）tetr 可以阻止四环素进入细胞。 （3）camr 生成氯霉素羟乙酰基衍生物，使之失去毒性。 （4）neor（kanr）氨基糖苷磷酸转移酶使G418（卡那霉素衍生物）失活 （5）hygr 使潮霉素β失活。 第三步：看多克隆位点（MCS）。它具有多个限制酶的单一切点。便于外源基因的插入。如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，而便于筛选。决定能不能放目的基因以及如何放置目的基因。 第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外源DNA片段。一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。 第五步：是否含有表达系统元件，即启动子－核糖体结合位点－克隆位点－转录终止信号。这是用来区别克隆载体与表达载体。克隆载体中加入一些与表达调控有关的元件即成为表达载体。选用那种载体，还是要以实验目的为准绳。 启动子－核糖体结合位点－克隆位点－转录终止信号 ＃启动子－促进DNA转录的DNA顺序，这个DNA区域常在基因或操纵子编码顺序的上游，是DNA分子上可以与RNApol特异性结合并使之开始转录的部位，但启动子本身不被转录。 ＃增强子/沉默子－为真核基因组（包括真核病毒基因组）中的一种具有增强邻近基因转录过程的调控顺序。其作用与增强子所在的位置或方向无关。即在所调控基因上游或下游均可发挥作用。/沉默子－负增强子，负调控序列。 ＃核糖体结合位点/起始密码/SD序列（Rbs/AGU/SDs）：mRNA有核糖体的两个结合位点，对于原核而言是AUG（起始密码）和SD序列。l ＃转录终止顺序（终止子）/翻译终止密码子：结构基因的最后一个外显子中有一个AATAAA的保守序列，此位点down－stream有一段GT或T富丰区，这2部分共同构成poly（A）加尾信号。
所有质粒载体汇总
酿酒酵母表达载体 pYES2,pYES2/NT,pYES2/CT,pYES3,pYES6, pYCplac22-GFP, 酵母载体pAUR123,pRS303TEF,pRS304, pRS305,pRS306,pY13TEF,pY14TEF pY15TEF, pY16TEF, 酵母基因重组表达载体pUG6, pSH47, 酵母单杂载体pHISi,pLacZi,pHIS2, pGAD424,酵母双杂交系统：酿酒酵母Y187, 酿酒酵母AH109;质粒pGADT7,pGBKT7 ;对照质粒pGBKT7-53 , pGBKT7-lam , pGADT7-T , PCL1，酿酒酵母菌株INVSc1,YM4271, AH109,丫187,丫190, 毕赤酵母表达载体 pPIC9K,pPIC9K-His,pPIC3.5K,pPICZalphaA,B,C,pPICZA,B,C,pGAPZ a A,pAO815,pPIC9k-His,pHIL-S1,pPink hc , 配套毕赤酵母Pichiapink, 毕赤酵母宿主X33, KM71 , KM71H , GS115, 原核表达载体pQE30,31,32,40,60,61,62等原核表达载体，包括pET系列，pET-GST, pGEX 系列(含GST标签)，pMAL 系列pMAL-c2x,-c4x,-c4e,-c5x,- p5x,pBAD,pBADHis,pBADmycHis 系列，pQE 系列,pTrc99a,pTrcHis系列， pBV220,221,222,pTXB 系列，pLLP-ompA,pIN-III-ompA (分泌型表达系列),pQBI63 (原核表达带荧光)pET3a, pET 3d, pET 11a, pET 12a, pET 14b, pET 15b, pET 16b, pET 17b, pET 19b, pET 20b, pET 21a,b,d, pET 22b, pET 23a, pET 23b, pET 24a,b, pET 25b, pET 26b, pET 27b, pET 28a,b, pET 29a, pET 30a, pET 31b, pET 32a, pET 35b, pET 38b, pET 39b, pET 40b, pET 41a,b pET 42a, pET 43.1a,b pET 44a, pET 49b pET302,303 pET His,pET Dsb,pET GST,pET Trx pQE2, pQE9 pQE30,31,32, pQE 40 pQE70 pQE80L pQETirs system pRSET-A pRSET-B pRSET-C pGEX4T-1,-2,-3,5x-1,6p-1,6p-2,2tk,3c pBV220,221,222 pTrcHisA,B,C pBAD24,34,43 pBAD HisA,B,C pPi nPoi nt-Xa1,Xa2,Xa3 pMALc2x, p2x pBV220 pGEM Ex1, pGEM7ZF (+) , pTrc99A, pTwin1, pEZZ18 pkk232-8,pkk 233- 3,pACYC184,pBR322,pUC119 pTYB1,pTYB2,pTYB4,pTYB11 pBlueScript SK (+) ,pBlueScript SK (-) pLLP ompA, pINIIIompA, pMBP-P ,pMBP-C,大肠杆菌冷激质粒：pColdI pColdII pColdIII pColdTF原核共表达质粒：pACYCduet-1,pETduet- 1,pCDFduet-1, pRSFduet-1 Takara公司大肠杆菌分子伴侣：pG-KJE8 pGro7 pKJE7 pGTf2 pTf16 大肠杆菌宿主细胞：DH5a JM101 JM103
质粒图谱的阅读方法
---------------------------------------------------------------最新资料推荐------------------------------------------------------ 质粒图谱的阅读方法 质粒图谱的阅读方法载体主要有病毒和非病毒两大类,其中质粒 DNA 是一种新的非病毒转基因载体。 一、一个合格质粒的组成要素 a. 复制起始位点 Ori 即控制复制起始的位点。 原核生物 DNA分子中只有一个复制起始点。 而真核生物 DNA分子有多个复制起始位点。 b. 抗生素抗性基因可以便于加以检测，如 Amp+ ,Kan+ c. 多克隆位点 MCS 克隆携带外源基因片段 d. P/E 启动子/增强子 e. Terms 终止信号 f. 加 poly（A）信号可以起到稳定 mRNA 作用二、如何阅读质粒图谱第一步： 首先看 Ori 的位置，了解质粒的类型（原核/真核/穿梭质粒）。 第二步： 再看筛选标记，如抗性，决定使用什么筛选标记。 （1） Ampr 水解－内酰胺环，解除氨苄的毒性。 （2） tetr 可以阻止四环素进入细胞。 （3） camr 生成氯霉素羟乙酰基衍生物，使之失去毒性。 （4） neor（kanr）氨基糖苷磷酸转移酶使 G418（长那霉素衍生物）失活（5） hygr 使潮霉素失活。 第三步： 1 / 6
看多克隆位点（MCS）。 它具有多个限制酶的单一切点。 便于外源基因的插入。 如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，而便于筛选。 决定能不能放目的基因以及如何放置目的基因。 第四步： 再看外源 DNA 插入片段大小。 质粒一般只能容纳小于 10Kb 的外源 DNA 片段。 一般来说，外源DNA 片段越长，越难插入，越不稳定，转化效率越低。 第五步： 是否含有表达系统元件，即启动子－核糖体结合位点－克隆位点－转录终止信号。 这是用来区别克隆载体与表达载体。 克隆载体中加入一些与表达调控有关的元件即成为表达载体。 选用那种载体，还是要以实验目的为准绳。 启动子－核糖体结合位点－克隆位点－转录终止信号 a. 启动子－促进 DNA 转录的 DNA 顺序，这个 DNA 区域常在基因或操纵子编码顺序的上游，是 DNA 分子上可以与 RNApol 特异性结合并使之开始转录的部位，但启动子本身不被转录。 b. 增强子/沉默子－为真核基因组（包括真核病毒基因组）
质粒绘图的专业软件——Winplas 使用说明
质粒绘图的专业软件——Winplas 2.7使用说明 作者：佚名来源：生物秀时间：2008-6-27 实验仪器大全实验试剂大全 Winplas作为一个质粒绘图的专业软件，功能强大，而且极易上手，它可以绘制出具有发表质量的质粒图谱。可广范应用于论文、教程的质粒插图，它的特性包括： 1，无论是否知道质粒的原始序列都能绘制质粒图，像Vector NTI等综合软件也能绘制质粒图，但有一个前提就是首先得知道质粒得原始序列； 2，可读入各种流行得序列格式文件，能方便地导入各种序列信息； 3，可自动在识别序列中的限制性酶切位点； 4，可对序列进行各种编辑，如：从文件插入序列、置换序列、序列编辑、部分序列删除等 5，绘图功能强大，如：位点标签、任意位置文字插入、生成彩图、线性或环形质粒图谱，可输出到剪贴板或图像文件。 软件下载地址：https://www.wendangku.net/doc/7a17902423.html,/Soft/2008/2571.htm 一，在不知道序列结构时绘制质粒图： 1，点击File菜单中地New命令，出现一个MapView窗口，同时工具栏中地绘图命令显亮。 2，点击“Insert”菜单中的“Blank Seqment”命令，出现一个“Create New Plasmid”对话框。 －在Title栏中填入质粒名称，如pUC18。 －在Base Pairs栏中填入质粒大小，如3000。 －在Type单选框中设制质粒图谱为线型还是环形。 3，点击OK后，在Map View窗口就会出现一个圆环，其中有质粒名称及质粒大小。 4，下面的工作就是向圆环上添加文字描述、标记及弧。 ①点击“Insert”菜单中的“Text”命令，或直接点击工具栏中的“Text”按纽，出现“Edit Text object”对话框。 －在“Text”书签中填入“Text”的内容，如：This is PUB 18’s map，选择左右对齐及居中。 －选定相应字符可以加黒、斜体等。 －在Font书签中改变字体格式、大小及颜色。 －文字就出现在Map View窗口中，使用鼠标左健，就可以随意拖动其位置。 ②点击“Insert”菜单中“Maker”命令或直接点击工具栏中的“Maker”按钮，出现”Edit Maker Object”对话框。 －此时，在Maker书签中的Base Pair填入添加Maker的位置，如800。 －在Label栏中，填入Maker的名称，如HindⅢ。同样可以改变HindⅢ的字体样式：加黑，斜体等。 －同样在Font书签中可以改变字体的样式、大小及颜色。 －点击OK，则在圆环800bp的相应位置出现HindⅢ的标记。 ③点击“Insert”菜单中“Arc”命令，弹出”Edit Arc Object”对话框。 －在Arc书签中的Base Pair栏中，填入Arc的起始位置，如1000。 －在Length栏中填入Arc的长度，如400。在Label栏中填入Arc的名称，如Amp。 －在Arc Style书签中，首先确定该Arc是否有箭头以及箭头的方向。分别对应none, 5’→3’,3’→5’,及双向。（生物秀-专心做生物！https://www.wendangku.net/doc/7a17902423.html,） －通过Width来确定Arc的宽度
阅读质粒图谱的基本方法
阅读质粒图谱的基本方法.txt爱情是彩色气球，无论颜色如何严厉，经不起针尖轻轻一刺。一流的爱人，既能让女人爱一辈子，又能一辈子爱一个女人！[转帖] 阅读质粒图谱的基本方法 载体主要有病毒和非病毒两大类,其中质粒DNA是一种新的非病毒转基因载体。一、一个合格质粒的组成要素 a复制起始位点Ori 即控制复制起始的位点。原核生物DNA分子中只有一 个复制起始点。而真核生物DNA分子有多个复制起始位点。 b 抗生素抗性基因可以便于加以检测，如Amp ,Kan c 多克隆位点MCS 克隆携带外源基因片段 d P/E 启动子/增强子 e Terms 终止信号 f 加poly（A）信号可以起到稳定mRNA作用 二、如何阅读质粒图谱 第一步：首先看Ori的位置，了解质粒的类型（原核/真核/穿梭质粒） 第二步：再看筛选标记，如抗性，决定使用什么筛选标记。 （1）Ampr 水解β－内酰胺环，解除氨苄的毒性。 （2）tetr 可以阻止四环素进入细胞。 （3）camr 生成氯霉素羟乙酰基衍生物，使之失去毒性。 （4）neor（kanr）氨基糖苷磷酸转移酶使G418（长那霉素衍生物）失活 （5）hygr 使潮霉素β失活。 第三步：看多克隆位点（MCS）。它具有多个限制酶的单一切点。便于 外源基因的插入。如果在这些位点外有外源基因的插入，会导致某种标 志基因的失活，而便于筛选。决定能不能放目的基因以及如何放置目的 基因。 第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外 源DNA片段。一般来说，外源DNA片段越长，越难插入，越不稳定，转 化效率越低。 第五步：是否含有表达系统元件，即启动子－核糖体结合位点－克隆位 点－转录终止信号。这是用来区别克隆载体与表达载体。克隆载体中加入 一些与表达调控有关的元件即成为表达载体。选用那种载体，还是要 以实验目的为准绳。 启动子－核糖体结合位点－克隆位点－转录终止信号 a 启动子－促进DNA转录的DNA顺序，这个DNA区域常在基因或操纵子 编码顺序的上游，是DNA分子上可以与RNApol特异性结合并使之开始转 录的部位，但启动子本身不被转录。 b增强子/沉默子－为真核基因组（包括真核病毒基因组）中的一种具有 增强邻近基因转录过程的调控顺序。其作用与增强子所在的位置或方向 无关。即在所调控基因上游或下游均可发挥作用。/沉默子－负增强子， 负调控序列。 c核糖体结合位点/起始密码/SD序列（Rbs/AGU/SDs）：mRNA有核糖体
如何阅读质粒图谱
一、载体主有病毒和非病毒两大类，其中质粒DNA是一种新的非病毒转基因载体。 一、一个合格质粒的组成要素 复制起始位点Ori，即控制复制起始的位点。原核生物DNA分子中只有一个复制起始点。而真核生物DNA分子有多个复制起始位点。抗生素抗性基因：可以便于加以检测，如Amp+ ，Kan+ 多 克隆位点：MCS克隆携带外源基因片段 P/E：启动子/增强子 Terms：终止信号 加poly（A）信号：可以起到稳定mRNA作用 二、如何阅读质粒图谱 第一步：首先看Ori的位置，了解质粒的类型（原核/真核/穿梭质粒） Ori的箭头指复制方向，其他元件标注的箭头多指转录方向（正向）。 第二步：再看筛选标记，如抗性，决定使用什么筛选标记： （1）Ampr：水解β-内酰胺环，解除氨苄的毒性。 （2）tetr ：可以阻止四环素进入细胞。 （3）camr：生成氯霉素羟乙酰基衍生物，使之失去毒性。 （4）neor（kanr）：氨基糖苷磷酸转移酶，使G418（卡那霉素衍生物）失活。 （5）hygr：使潮霉素β失活。 第三步：看多克隆位点（MCS）。它具有多个限制酶的单一切点，便于外源基因的插入。如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，而便于筛选。决定能不能放目的基因以及如何放置目的基因。 第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外源DNA片段。一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。 第五步：是否含有表达系统元件，即启动子－核糖体结合位点－克隆位点－转录终止信号。这是用来区别克隆载体与表达载体。克隆
载体中加入一些与表达调控有关的元件即成为表达载体。选用那种载体，还是要以实验目的为准绳。 相关概念： 启动子－核糖体结合位点－克隆位点－转录终止信号 启动子－促进DNA转录的DNA顺序，这个DNA区域常在基因或 操纵子编码顺序的上游，是DNA分子上可以与RNApol特异性结合并使之开始转录的部位，但启动子本身不被转录。 增强子/沉默子－为真核λ基因组（包括真核病毒基因组）中的一种具有增强邻近基因转录过程的调控顺序。其作用与增强子所在的位置或方向无关。即在所调控基因上游或下游均可发挥作用。沉默子－负增强子，负调控序列。 核糖体结合位点/起始密码/SD序列（Rbs/AGU/SDs）：mRNA有核糖体的两个结合位点，对于原核而言是AUG（起始密码）和SD序列。 λ转录终止顺序（终止子）/翻译终止密码子：结构基因的最后一个外显子中有一个AATAAA的保守序列，此位点down－stream有一段GT 或T富丰区，这2部分共同构成poly（A）加尾信号。结构基因的最后一个外显子中有一个AATAAA的保守序列，此位点down－stream有一段GT或T富丰区，这2部分共同构成poly（A）加尾信号。 三、载体及其分类 载体：即要把一个有用的基因（目的基因——研究或应用基因）通过基因工程手段送到生物细胞（受体细胞），需要运载工具（交通工具）携带外源基因进入受体细胞，这种运载工具就叫做载体（vector）。 P.S.基因工程所用的vector实际上是DNA分子，是用来携带目的 基因片段进入受体细胞的DNA。 载体的分类 按功能分成：（1）克隆载体：都有一个松弛的复制子，能带动 外源基因，在宿主细胞中复制扩增。它是用来克隆和扩增DNA片段（基因）的载体。（所以有时实验时扩增效率低下，要注意是不是使用的严谨型载体）（2）表达载体：具有克隆载体的基本元件 （ori,Ampr,Mcs等）还具有转录/翻译所必需的DNA顺序的载体。
怎么查找质粒图谱
经常在坛子里看到一些人求助质粒图谱，很多时候我发现其实有些质粒图谱还是很容易找到了，刚开始帮忙查找了下，还公布了一些查找质粒图谱比较好的网站，后来看得多了，很多时候，这样的帖子直接跳过了。今天又看到几个求质粒图谱的帖子，因此决定就查找质粒图谱的方法，写个总结帖子，希望对虫子们有些帮助。这些方法，大部分是自己学习的过程中积累的，也许总结得还不够全面，望其他虫友指正。 方法一：安装软件Vector NT 做分子实验，经常和不同的质粒打交道，了解各种质粒的图谱信息是必需的，invitrogen公司的这款软件绝对是分子生物学虫子们的福音，功能强大、界面美观，使用起来很人性化。后面的很多方法都是基于在这款软件的使用之上，因此个人觉得要想对质粒图谱了解更直观，安装这款软件是非常必要的。而且，一旦安装了这款软件，你就发现这款软件的软件包里面会包括invitrogen公司的所有质粒图谱信息和其他比较常见和经典的质粒图谱（不是有虫子求pRS系列质粒吗？帖子链接https://www.wendangku.net/doc/7a17902423.html,/bbs/viewthread.php?tid=3103223&fpage=1，如下图，数据库中本身就有很多）。这里就不一一细说，各位虫子可以自己体验下。（这款软件的下载和使用说明书站内很多）
方法二：查找质粒图谱的网站： 这个之前有人求助质粒图谱时，我在回应求助帖里面公布过几个我经常用的网址，估计不是专题，很多人没看到，现在在此重新总结下 1.Vector Database 地址：https://https://www.wendangku.net/doc/7a17902423.html,/g?a=vdb 这个网站很页面很人性化，直入主题，也是我经常用到一个网站，比如同样这个帖子求pRS类质粒图谱（注意，是一类质粒图谱，没关系，照样能找到），直接在搜索框输入pRS，可以看到，之类质粒一共有三十多个。
【Y2H操作方法-HXY整理】
实验步骤说明**： 1. 诱饵质粒pGBKT7-bait的构建 2.诱饵融合蛋白BD-bait对AH109酵母的自激活作用及毒性的检测【重要的前期准备】 3. 人胰腺cDNA文库的滴定和扩增【不用做了，直接用质粒】 4. 转化AH109酵母后转化子（阳性克隆）的筛选【顺序转化或共转化】 5. 蛋白质相互作用的验证【一对一验证】 ** 2.诱饵融合蛋白BD-bait对AH109酵母的自激活作用及毒性的检测 2.1 LacZ自激活的检测. 2.2 HIS3自激活的检测 2.3 融合蛋白对酵母菌的毒性检测 4. 文库转化AH109酵母后转化子的筛选 4.1 文库质粒转化pGBKT7-bait/AH109酵母 使用标准的PEG/LiAc转化法将文库质粒转化入已含有pGBKT7-bait质粒的AH109酵母，涂布在SD-Ade-His-Leu-Trp平板上进行重组子筛选 4.2 报告基因ADE2、HIS3和lacZ激活的验证 ADE2、HIS3的激活：酵母能够在SD-Ade-His-Leu-Trp平板生长 LacZ的激活：β-半乳糖苷酶显色反应呈蓝色 4.3 pACT2-cDNA质粒的获取： ①从酵母中抽提质粒，得到pGBKT7-bait和pACT2-cDNA的混合物
②转化E. coli，涂布在含Amp的LB平板上，由于质粒不相容性，转化子中便只含有pACT2-cDNA质粒 ③从转化成功的E. coli中抽提出pACT2-cDNA质粒 5. 蛋白质相互作用的验证【一对一验证】 5.1 由于酵母双杂交的假阳性很普遍，所以筛选得到相互作用蛋白的cDNA序列之后，还应将分离出 的各种pACT2-cDNA再次单独转化pGBKT7-bait/AH109酵母，即一对一验证，再次检测它们对报告基因的激活情况，排除假阳性 5.2 将pACT2-cDNA质粒送测序，得到cDNA片段的序列，在NCBI数据库Blast查出其对应的蛋白 质 5.3 另外，还必须分析这些pACT2-cDNA质粒的构建情况。由于商品文库构建的特殊性，要特别关注 这些插入的cDNA片段是否发生了frameshift 5.4 在上述验证都通过的情况下，仍然需要用诸如免疫共沉淀、GST-pull down、细胞共定位等实验来 对这些相互作用来进行验证 公司的这三个操作手册：
常用pGEX载体图谱
Rosetta系列的表达菌株可以提供T7 RNA聚合酶，它能表达PET系列载体上的外源基因。。。pGEX系列载体上的外源基因不需要T7 RNA聚合酶，普通的大肠杆菌经IPTG诱导即可表达 Tac启动子是一组由Lac和trp启动子人工构建的杂合启动子，受Lac阻遏蛋白的负调节，它的启动能力比Lac和trp都强。其中Tac 1是由Trp启动子的－35区加上一个合成的46 bp DNA片段(包括Pribnow 盒)和Lac操纵基因构成，Tac 12是由Trp的启动子-35区和Lac启动子的-10区，加上Lac操纵子中的操纵基因部分和SD序列融合而成 蛋白标签： A myc tag is a polypeptide protein tag derived from the c-myc gene product that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits. A myc tag can be used in many different assays that require recognition by an antibody. If there is no antibody against the studied protein, adding a myc-tag allows one to follow the protein with an antibody against the Myc epitope. Examples are cellular localization studies by immunofluorescence or detection by Western blotting. The peptide sequence of the myc-tag is (in 1- and 3-letter codes, respectively): N-EQKLISEEDL-C, N-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-C, where N stands for Amino-terminus and C stands for Carboxy terminus. The tag is approximately 1202 Daltons in atomic mass and has 10 amino acids. It can be fused to the C-terminus and the N-terminus of a protein. It is advisable not to fuse the tag directly behind the signal peptide of a secretory protein, since it can interfere with translocation into the secretory pathway. A monoclonal antibody against the myc epitope, named 9E10, is available from the non-commercial Developmental Studies Hybridoma Bank
如何阅读分析质粒图谱
基因酷质粒图谱https://www.wendangku.net/doc/7a17902423.html,/bbs/forum-38-1.html，收藏了将近800种质粒的图谱及相关信息 特向大家推荐，介绍及使用方法见： https://www.wendangku.net/doc/7a17902423.html,/bbs/thread-417-1-1.html 质粒图谱信息 一.九种表达载体 Pllp-OmpA, pllp-STII, pMBP-P, pMBP-C, pET-GST, pET-Trx, pET-His, pET-CKS, pET-DsbA 二.克隆载体 pTZ19R DNA pUC57 DNA PMD18T PQE30 pUC18 pUC19 pTrcHisA pTrxFus pRSET-A pRSET-B pVAX1 PBR322 pbv220 pBluescript II KS (+) L4440 pCAMBIA-1301 pMAL-p2X pGD926 三.PET系列表达载体 Protein Expression ? Prokaryotic Expression ? pET Dsb Fusion Systems 39b and 40b Protein Expression ? Prokaryotic Expression ? pET Expression System 33b Protein Expression ? Prokaryotic Expression ? pET Expression Systems Protein Expression ? Prokaryotic Expression ? pET Expression Systems plus Competent Cells Protein Expression ? Prokaryotic Expression ? pET GST Fusion Systems 41 and 42 Protein Expression ? Prokaryotic Expression ? pET NusA Fusion Systems 43.1 and 44 Protein Expression ? Prokaryotic Expression ? pET Vector DNA Protein Purification ? Purification Systems ? Strep?Tactin Resins and Purification Kits 四.PGEX系列表达载体
☆如何查找质粒图谱
如何查找质粒图谱 编者小木虫论坛susizheng 经常在坛子里看到一些人求助质粒图谱，很多时候我发现其实有些质粒图谱还是很容易找到了，刚开始帮忙查找了下，还公布了一些查找质粒图谱比较好的网站，后来看得多了，很多时候，这样的帖子直接跳过了。今天又看到几个求质粒图谱的帖子，突发奇想，就查找质粒图谱的方法，写个总结帖子吧，希望对虫子们有些帮助。这些方法，大部分是自己学习的过程中偶尔发现，也许总结得还不够全面，望其他虫友指正。 方法一：安装软件Vector NT 做分子实验，经常和不同的质粒打交道，了解各种质粒的图谱信息是必需的，invitrogen公司的这款软件绝对是分子生物学虫子们的福音，功能强大、界面美观，使用起来很人性化。后面的很多方法都是基于在这款软件的使用之上，因此个人觉得要想对质粒图谱了解更直观，安装这款软件是非常必要的。而且，一旦安装了这款软件，你就发现这款软件的软件包里面会包括invitrogen公司的所有质粒图谱信息和其他比较常见和经典的质粒图谱（如下图，不是有虫子求pRS 系列质粒吗？如下图，数据库中本身就有很多）。这里就不一一细说，各位虫子可以自己体验下。（这框软件的下载和使用说明书站内很多） 方法二：查找质粒图谱的网站： 这个之前有人求助质粒图谱时，我在回应求助帖里面公布过几个我经常用的网址，估计不是专题，很多人没看到，现在在此重新总结下。 1.Vector Database 地址：https://https://www.wendangku.net/doc/7a17902423.html,/g?a=vdb 这个网站很页面很人性化，直入主题，也是我经常用到一个网站，比如这个帖子https://www.wendangku.net/doc/7a17902423.html,/bbs/viewthread.php?tid=3103223&fpage=1，这个虫友求pRS类质
质粒载体分类及阅读
质粒载体分类及阅读 一.九种表达载体 Pllp-OmpA, pllp-STII, pMBP-P, pMBP-C, pET-GST, pET-Trx, pET-His, pET-CKS, pET-DsbA 二.克隆载体 pTZ19R DNA pUC57 DNA PMD18T PQE30 pUC18 pUC19 pTrcHisA pTrxFus pRSET-A pRSET-B pVAX1 PBR322 pbv220 pBluescript II KS (+) L4440 pCAMBIA-1301 pMAL-p2X pGD926 三.PET系列表达载体 Protein Expression ? Prokaryotic Expression ? pET Dsb Fusion Systems 39b and 40b Protein Expression ? Prokaryotic Expression ? pET Expression System 33b Protein Expression ? Prokaryotic Expression ? p ET Expression Systems Protein Expression ? Prokaryotic Expression ? pET Expression Systems plus Competent Cells Protein Expression ? Prokaryotic Expression ? pET GST Fusion Systems 41 and 42 Protein Expression ? Prokaryotic Expression ? pET NusA Fusion Systems 43.1 and 44 Protein Expression ? Prokaryotic Expression ? pET Vector DNA Protein Purification ? Purification Systems ? Strep?Tactin Resins and Purification Kits 四.PGEX系列表达载体 T EcoR pGEX-1 I/BAP pGEX-2T pGEX-2TK pGEX-3X
查询质粒方法汇总
如何查找质粒图谱之我见——plasmid map, Vector Sequence方法汇总 经常在坛子里看到一些人求助质粒图谱，很多时候我发现其实有些质粒图谱还是很容易找到了，刚开始帮忙查找了下，还公布了一些查找质粒图谱比较好的网站，后来看得多了，很多时候，这样的帖子直接跳过了。今天又看到几个求质粒图谱的帖子，因此决定就查找质粒图谱的方法，写个总结帖子，希望对虫子们有些帮助。这些方法，大部分是自己学习的过程中积累的，也许总结得还不够全面，望其他虫友指正。 方法一：安装软件Vector NT 做分子实验，经常和不同的质粒打交道，了解各种质粒的图谱信息是必需的，invitrogen公司的这款软件绝对是分子生物学虫子们的福音，功能强大、界面美观，使用起来很人性化。后面的很多方法都是基于在这款软件的使用之上，因此个人觉得要想对质粒图谱了解更直观，安装这款软件是非常必要的。而且，一旦安装了这款软件，你就发现这款软件的软件包里面会包括invitrogen公司的所有质粒图谱信息和其他比较常见和经典的质粒图谱。这里就不一一细说，各位虫子可以自己体验下。（这款软件的下载和使用说明书站内很多） 方法二：查找质粒图谱的网站： 这个之前有人求助质粒图谱时，我在回应求助帖里面公布过几个我经常用的网址，估计不是专题，很多人没看到，现在在此重新总结下 1.Vector Database 地址：https://https://www.wendangku.net/doc/7a17902423.html,/g?a=vdb 这个网站很页面很人性化，直入主题，也是我经常用到一个网站，比如同样这个帖子求pRS类质粒图谱（注意，是一类质粒图谱，没关系，照样能找到），直接在搜索框输入pRS，可以看到，之类质粒一共有三十多个。
认识质粒图谱
一、如何阅读质粒图谱 载体主要有病毒和非病毒两大类，其中质粒DNA是一种新的非病毒转基因载体。 一、一个合格质粒的组成要素 复制起始位点Ori，即控制复制起始的位点。原核生物DNA分子中只有一个复制起始点。 而真核生物DNA分子有多个复制起始位点。 抗生素抗性基因：可以便于加以检测，如Amp+ ，Kan+ 多l克隆位点：MCS克隆携带外源基因片段 P/E：启动子/增强子 Terms：终止信号 加poly（A）信号：可以起到稳定mRNA作用 二、如何阅读质粒图谱 第一步：首先看Ori的位置，了解质粒的类型（原核/真核/穿梭质粒） Ori的箭头指复制方向，其他元件标注的箭头多指转录方向（正向）。 第二步：再看筛选标记，如抗性，决定使用什么筛选标记： （1）Ampr：水解β-内酰胺环，解除氨苄的毒性。 （2）tetr ：可以阻止四环素进入细胞。 （3）camr：生成氯霉素羟乙酰基衍生物，使之失去毒性。 （4）neor（kanr）：氨基糖苷磷酸转移酶，使G418（卡那霉素衍生物）失活。 （5）hygr：使潮霉素β失活。 第三步：看多克隆位点（MCS）。它具有多个限制酶的单一切点，便于外源基因的插入。 如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，而便于筛选。决定能不能放目的基因以及如何放置目的基因。 第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外源DNA片段。 一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。 第五步：是否含有表达系统元件，即启动子－核糖体结合位点－克隆位点－转录终止信号。 这是用来区别克隆载体与表达载体。克隆载体中加入一些与表达调控有关的元件即成为表达载体。选用那种载体，还是要以实验目的为准绳。 二、相关概念： 启动子－核糖体结合位点－克隆位点－转录终止信号 启动子－促进DNA转录的DNA顺序，这个DNA区域常在基因或操纵子编码顺序的上游，是DNA分子上可以与RNApol特异性结合并使之开始转录的部位，但启动子本身不被转录。 增强子/沉默子－为真核l基因组（包括真核病毒基因组）中的一种具有增强邻近基因转录过程的调控顺序。其作用与增强子所在的位置或方向无关。即在所调控基因上游或下游均可发挥作用。沉默子－负增强子，负调控序列。 核糖体结合位点/起始密码/SD序列（Rbs/AGU/SDs）：mRNA有核糖体的两个结合位点，对于原核而言是AUG（起始密码）和SD序列。 l转录终止顺序（终止子）/翻译终止密码子：结构基因的最后一个外显子中有一个AATAAA 的保守序列，此位点down－stream有一段GT或T富丰区，这2部分共同构成poly（A）加尾信号。结构基因的最后一个外显子中有一个AATAAA的保守序列，此位点down－stream有一段GT或T富丰区，这2部分共同构成poly（A）加尾信号。 三、载体及其分类 载体：即要把一个有用的基因（目的基因——研究或应用基因）通过基因工程手段送到生物细胞（受体细胞），需要运载工具（交通工具）携带外源基因进入受体细胞，这种运载