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=== Choosing A Plasmid Backbone === | |||
When choosing what plasmid backbone to use, you have many elements to consider. Here is a [https://www.addgene.org/empty-backbones/ guide to Addgene's empty vector backbones]. For the most part, we will assume that you want to express a gene; however, we have a section at the end for if you are studying a different genetic element or want to express shRNA. | |||
<html> | |||
<section id="host"> | |||
<h2>Species-Specific Expression</h2> | |||
<p>If you want to drive expression of your favorite gene, you will need | |||
a plasmid with a promoter that will be functional in your host | |||
organism.</p> | |||
<table class="empty-backbone-table table table-striped table-hover"> | |||
<thead> | |||
<tr> | |||
<th>Host</th> | |||
<th>Relevant Promoters</th> | |||
<th>Representative Empty Backbones</th> | |||
</tr> | |||
</thead> | |||
<tbody> | |||
<tr> | |||
<td>Mammalian</td> | |||
<td>CMV, SV40, EF1a, CAG</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/10791/">pSG5L Flag HA</a> - Transient | |||
expression</li> | |||
<li><a href="http://www.addgene.org/1764/">pBABE-puro</a> - Retroviral | |||
expression</li> | |||
<li><a href="http://www.addgene.org/12257/">pWPXL</a> - Lentiviral | |||
expression</li> | |||
<li><a href="http://www.addgene.org/16542/">pBI-MCS-EGFP</a> - | |||
Tet-inducible</li> | |||
<li><a href="http://www.addgene.org/browse/article/4218/">Transient | |||
expression</a> - Gradia Lab plasmids for mammalian | |||
expression, Ligation Independent Cloning (LIC)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Bacteria</td> | |||
<td>Lac, T7, araBAD</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/37501/">pBAD LIC cloning vector</a> - | |||
Bacterial expression (see <a href= | |||
"/Scott_Gradia/">all expression vectors</a> from | |||
the Gradia lab)</li> | |||
<li><a href="http://www.addgene.org/26092/">p15TV-L</a> - His-tagged | |||
bacterial expression vector (see <a href= | |||
"/sgc/">tagged expression vectors</a> from the | |||
SGC)</li> | |||
<li><a href="http://www.addgene.org/17806/">pPro18</a> - | |||
Propionate-inducible expression vector (see entire | |||
<a href="http://www.addgene.org/browse/article/2026/">pPro | |||
collection</a>)</li> | |||
<li><a href="http://www.addgene.org/browse/article/3609/">Arrowsmith Lab | |||
Plasmids</a> - More LIC bacterial cloning | |||
vectors</li> | |||
<li><a href="http://www.addgene.org/browse/article/6833/">pTD plasmid | |||
series</a> - Expression with Strep, His or | |||
optimized YFP tags for purification or localization | |||
experiments</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Yeast</td> | |||
<td>GAL4, PGK, ADH1, ADE2, TRP1</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/yeast-gateway/">Gateway destination | |||
vectors</a> - Inducible expression, fusion | |||
proteins, and more</li> | |||
<li><a href="http://www.addgene.org/browse/article/3784/">Sandmeyer Lab | |||
Plasmids</a> - Set of yeast expression vectors with | |||
various markers, promoters, etc</li> | |||
<li><a href="http://www.addgene.org/11259/">pRS420</a> - Yeast expression | |||
vector (see <a href="http://www.addgene.org/browse/article/509/">Cross | |||
lab collection</a> for more)</li> | |||
<li><a href="http://www.addgene.org/11342/">pLexA</a> and <a href= | |||
"/11343/">pACT2.2</a> - Yeast 2-hybrid | |||
plasmids</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Fly</td> | |||
<td>UAS, MT</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/browse/article/3631/">Targeted Gene | |||
Expression</a> - Rubin lab plasmids for drosophila | |||
expression</li> | |||
<li><a href="http://www.addgene.org/17923/">pMT-puro</a> - Inducible gene | |||
expression under the metallothionein promoter</li> | |||
<li><a href="http://www.addgene.org/31223/">pACU2</a> - Modified pUAST | |||
vector containing both P-elements and attB site for | |||
high expression of UAS-driven transgenes</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Worm</td> | |||
<td>unc-54, variety of worm gene promoters</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/firelab/">C. elegans vector kit</a> - | |||
Collection of plasmids from Dr. Andrew Fire</li> | |||
<li><a href="http://www.addgene.org/1654/">L4440</a> - RNAi in C. | |||
elegans</li> | |||
<li><a href="http://www.addgene.org/browse/article/3655/">Dupuy Lab</a> | |||
and <a href="http://www.addgene.org/browse/article/5402/">Lehner Lab</a> | |||
vectors - Tools for nematode transgenesis</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Insect / Baculovirus</td> | |||
<td>Polyhedrin</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/browse/article/4215/">pFastBac LIC</a> and <a href= | |||
"/browse/article/4217/">pFastBac Dual LIC</a> - | |||
Vectors from the Gradia lab</li> | |||
<li><a href="http://www.addgene.org/26108/">pFB-LIC-Bse</a> - Insect | |||
expression vector from Gileadi lab</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Xenopus</td> | |||
<td>SP6 for transcription and injection</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/17095/">pCS2P+</a> - Transcription | |||
from the SP6 promoter</li> | |||
<li><a href="http://www.addgene.org/15681/">pCS2 mt-GFP</a> - Create | |||
myc-tag, GFP-tag fusions for transcription</li> | |||
<li><a href="http://www.addgene.org/22423/">pCSDest</a> - Easy cloning of | |||
your gene using Gateway recombination</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
</tbody> | |||
</table><a href="#top">Back to Top</a> | |||
</section> | |||
<section id="tag"> | |||
<h2>Epitope Tag or Fusion Protein</h2> | |||
<p>Tags and fusion proteins are excellent tools for further | |||
understanding the function of your favorite gene. For example, fusing | |||
your protein to an epitope tag, such as Flag or HA, will allow you to | |||
easily identify your protein using an antibody against that epitope. | |||
This could allow you to conduct western blots or immunoprecipitations | |||
of your favorite gene even if you do not have an antibody against it. | |||
Another common scenario is fusing your protein to another protein, such | |||
as GFP, which allows you to visualize the cellular localization of your | |||
protein.</p> | |||
<p>Just remember that when you are designing your plasmid you should | |||
keep your gene "in frame" with the fusion protein. This means that the | |||
final product should be translated as a single string of amino acids | |||
that preserves the sequence of your gene and of the fusion protein.</p> | |||
<table class="empty-backbone-table table table-striped table-hover"> | |||
<thead> | |||
<tr> | |||
<th>Tag or Fusion | |||
Protein</th> | |||
<th>Common uses</th> | |||
<th>Representative Empty | |||
Backbones</th> | |||
</tr> | |||
</thead> | |||
<tbody> | |||
<tr> | |||
<td>Flag</td> | |||
<td>Epitope tag</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/10792/">pcDNA3 Flag HA</a> - | |||
N-terminal Flag-HA for mammalian expression</li> | |||
<li><a href="http://www.addgene.org/26105/">pNIC-CTHF</a> - C-terminal | |||
TEV-His6-Flag for bacterial expression</li> | |||
<li><a href="http://www.addgene.org/29715/">pET Flag TEV</a> - N-terminal | |||
Flag-TEV for bacterial expression</li> | |||
<li><a href="http://www.addgene.org/16331/">pCS2FLAG</a> - N or C-terminal | |||
2xFLAG tag in pCS for a variety of systems, | |||
including Xenopus</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>HA</td> | |||
<td>Epitope Tag</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/12555/">pKH3</a> - N or C-terminal | |||
3xHA tag for mammalian expression</li> | |||
<li><a href="http://www.addgene.org/12517/">pHAHA</a> - N-terminal double | |||
HA tag for mammalian expression</li> | |||
<li><a href="http://www.addgene.org/16330/">pCS2HA</a> - N or C-terminal | |||
2xHA tag in pCS for a variety of systems, including | |||
Xenopus</li> | |||
<li><a href="http://www.addgene.org/14246/">pAG423GPD-ccdB-HA</a> - | |||
C-terminal 3xHA tag for yeast expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Myc</td> | |||
<td>Epitope Tag</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/19400/">pKMyc</a> - N-terminal Myc | |||
tag for mammalian expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>6xHis</td> | |||
<td>Epitope Tag</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/18701/">pEZYmyc-His</a> - C-terminal | |||
Myc-His tag for mammalian expression (Gateway)</li> | |||
<li><a href="http://www.addgene.org/sgc/">SGC plasmids</a> - A variety of | |||
His tagged bacterial expression vectors</li> | |||
<li><a href="http://www.addgene.org/29653/">pET His6 TEV</a> - N-terminal | |||
His6-TEV tag for bacterial expression</li> | |||
<li><a href="http://www.addgene.org/37236/">pET His6</a> - C-terminal | |||
His6 tag for bacterial expression</li> | |||
<li><a href="http://www.addgene.org/11518/">pDest-527</a>- N-terminal | |||
His6 tag for bacterial expression (Gateway)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>GST</td> | |||
<td>Protein Purification</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/22227/">pEBG</a> - N-terminal GST for | |||
mammalian expression</li> | |||
<li><a href="http://www.addgene.org/29655/">pET His6 GST TEV</a> - | |||
N-terminal His6-GST-TEV for bacterial | |||
expression</li> | |||
<li><a href="http://www.addgene.org/11520/">pDest-565</a> - N-terminal | |||
His6-GST for bacterial expression (Gateway)</li> | |||
<li><a href="http://www.addgene.org/8908/">pPS892</a> - N-terminal GST | |||
for yeast expression</li> | |||
<li><a href="http://www.addgene.org/31240/">pDM#834</a> - C-terminal GST | |||
for C elegans expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>GFP</td> | |||
<td>Localization</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/13031/">pcDNA3-EGFP</a> - C-terminal | |||
GFP for mammalian expression</li> | |||
<li><a href="http://www.addgene.org/30127/">pcDNA3 GFP LIC cloning | |||
vector</a> - C-terminal GFP for mammalian | |||
expression</li> | |||
<li><a href="http://www.addgene.org/8856/">pPS808</a> - N-terminal GFP | |||
for yeast expression</li> | |||
<li><a href="http://www.addgene.org/29772/">pET GFP</a> - C-terminal GFP | |||
for bacterial expression</li> | |||
<li><a href="http://www.addgene.org/1494/">pPD95_75</a> - C-terminal GFP | |||
for C elegans expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Other Fluorescent Proteins</td> | |||
<td>Localization</td> | |||
<td>See full <a href="http://www.addgene.org/fluorescent-proteins/">Fluorescent | |||
Protein Guide</a></td> | |||
</tr> | |||
<tr> | |||
<td>NLS</td> | |||
<td>Nuclear Localization</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/20026/">pENTR4-myc-nuc</a> - pEntry | |||
vector that adds a C-terminal nuclear localization | |||
signal</li> | |||
<li><a href="http://www.addgene.org/45346/">FNpcDNA3</a> - N-terminal | |||
FLAG and SV40 nuclear localization signal</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Myr</td> | |||
<td>Membrane Localization</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/15300/">pWZL-Neo-Myr-Flag-DEST</a> - | |||
pDest vector that adds a N-terminal myristoylation | |||
signal</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
</tbody> | |||
</table> | |||
<p>For more information about tags, including amino acid sequences, see | |||
our <a href="http://www.addgene.org/mol_bio_reference/genetic_code/#tags">list of common | |||
tags</a>.</p><a href="#top">Back to Top</a> | |||
</section> | |||
<section id="selection"> | |||
<h2>Selectable Markers</h2> | |||
<p>Regardless of your delivery method, it's unlikely that all of your | |||
cells will take up your plasmid. Thus, many plasmids have markers on | |||
them so that you can find or select for only the cells that received | |||
the plasmid.</p> | |||
<table class="empty-backbone-table table table-striped table-hover"> | |||
<thead> | |||
<tr> | |||
<th>Selectable | |||
Marker</th> | |||
<th>Typical Host | |||
Organism</th> | |||
<th>Representative Empty | |||
Backbones</th> | |||
</tr> | |||
</thead> | |||
<tbody> | |||
<tr> | |||
<td>Neomycin (G418)</td> | |||
<td>Mammalian</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/1767/">pBABE-neo</a> - Retroviral | |||
gene expression</li> | |||
<li><a href="http://www.addgene.org/21916/">Tet-pLKO-neo</a> - | |||
Tet-inducible lentiviral shRNA expression</li> | |||
<li><a href="http://www.addgene.org/17392/">pLenti CMV Neo DEST</a> - | |||
Lentiviral Gateway destination vector for gene | |||
expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Puromycin</td> | |||
<td>Mammalian</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/1764/">pBABE-puro</a> - Retroviral | |||
gene expression</li> | |||
<li><a href="http://www.addgene.org/10878/">pLKO.1 - TRC cloning | |||
vector</a> - Lentiviral shRNA expression</li> | |||
<li><a href="http://www.addgene.org/21915/">Tet-pLKO-puro</a> - | |||
Tet-inducible lentiviral shRNA expression</li> | |||
<li><a href="http://www.addgene.org/8452/">pMKO.1 puro</a> - Retroviral | |||
shRNA expression</li> | |||
<li><a href="http://www.addgene.org/17452/">pLenti CMV Puro DEST</a> - | |||
Lentiviral Gateway destination vector for gene | |||
expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Hygromycin</td> | |||
<td>Mammalian</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/1765/">pBABE-hygro</a> - Retroviral | |||
gene expression</li> | |||
<li><a href="http://www.addgene.org/24150/">pLKO.1 hygro</a> - Lentiviral | |||
shRNA expression</li> | |||
<li><a href="http://www.addgene.org/21915/">Tet-pLKO-puro</a> - | |||
Tet-inducible lentiviral shRNA expression</li> | |||
<li><a href="http://www.addgene.org/17454/">pLenti CMV Hygro DEST</a> - | |||
Lentiviral Gateway destination vector for gene | |||
expression</li> | |||
<li><a href="http://www.addgene.org/17484/">pLenti CMV/TO Hygro</a> - | |||
Lentiviral Gateway destination vector for gene | |||
expression (tet inducible)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Zeocin/Bleo</td> | |||
<td>Mammalian</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/1766/">pBABE-zeo</a> - Retroviral | |||
gene expression</li> | |||
<li><a href="http://www.addgene.org/19277/">pLenti X1 GFP-Zeo DEST</a> - | |||
Lentiviral Gateway destination for shRNA | |||
expression</li> | |||
<li><a href="http://www.addgene.org/17294/">pLenti CMV/TO Zeo DEST</a> - | |||
Tet-inducible lentiviral Gateway destination vector | |||
for gene expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>GFP</td> | |||
<td>Varies</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/10668/">pBABE GFP</a> - Retroviral | |||
gene expression</li> | |||
<li><a href="http://www.addgene.org/10676/">pMKO.1 GFP</a> - Retroviral | |||
shRNA expression</li> | |||
<li><a href="http://www.addgene.org/12093/">pSico PGK GFP</a> - | |||
Conditional shRNA expression through lentiviral | |||
delivery</li> | |||
<li><a href="http://www.addgene.org/19732/">pLenti CMV GFP DEST</a> - | |||
Lentiviral Gateway destination vector for gene | |||
expression</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>URA3</td> | |||
<td>Yeast</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/26846/">pXP518</a> - Yeast expression | |||
vector</li> | |||
<li><a href="http://www.addgene.org/8856/">pPS808</a> - Yeast expression | |||
with N-terminal GFP</li> | |||
<li><a href="http://www.addgene.org/14139/">pAG306GAL-ccdB</a> - Yeast | |||
Gateway expression vector (see <a href= | |||
"/yeast-gateway/browse/">more</a> Gateway | |||
vectors)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>TRP1</td> | |||
<td>Yeast</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/26830/">pXP216</a> - Yeast expression | |||
vector (PGK1 promoter, 2μ)</li> | |||
<li><a href="http://www.addgene.org/26836/">pXP316</a> - Yeast expression | |||
vector (TEF1 promoter, CEN/ARS)</li> | |||
<li><a href="http://www.addgene.org/14135/">pAG304GAL-ccdB</a> - Yeast | |||
Gateway expression vector (see <a href= | |||
"/yeast-gateway/browse/">more</a> Gateway | |||
vectors)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>LEU2</td> | |||
<td>Yeast</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/26827/">pXP122</a> - Yeast expression | |||
vector (PGK1 promoter, CEN/ARS)</li> | |||
<li><a href="http://www.addgene.org/26833/">pXP222</a> - Yeast expression | |||
vector (PGK1 promoter, 2μ)</li> | |||
<li><a href="http://www.addgene.org/14137/">pAG305GAL-ccdB</a> - Yeast | |||
Gateway expression vector (see <a href= | |||
"/yeast-gateway/browse/">more</a> Gateway | |||
vectors)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>HIS3</td> | |||
<td>Yeast</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/26832/">pXP220</a> - Yeast expression | |||
vector (PGK1 promoter, 2μ)</li> | |||
<li><a href="http://www.addgene.org/14133/">pAG303GAL-ccdB</a> - Yeast | |||
Gateway expression vector (see <a href= | |||
"/yeast-gateway/browse/">more</a> Gateway | |||
vectors)</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
</tbody> | |||
</table><a href="#top">Back to Top</a> | |||
</section> | |||
<section id="viral"> | |||
<h2>Viral Expression and Packaging</h2> | |||
<p>Although transient expression is sufficient for some experiments, | |||
scientists often want to create stable cell lines in which the plasmid | |||
is incorporated into the host DNA. For mammalian cells, you can do this | |||
through viral delivery. <a href="http://www.addgene.org/viral-vectors/">Visit our viral vector page for more information.</a> Below are some common delivery methods:</p> | |||
<table class="empty-backbone-table table table-striped table-hover"> | |||
<tbody> | |||
<thead> | |||
<tr> | |||
<th>Delivery | |||
method</th> | |||
<th>Advantages</th> | |||
<th>Representative Empty | |||
Backbones</th> | |||
</tr> | |||
</thead> | |||
<tbody> | |||
<tr> | |||
<td>Lentiviral</td> | |||
<td>Can transduce both dividing and nondividing | |||
cells</td> | |||
<td>Addgene has an extensive collection of plasmids for | |||
packaging and expression. See our dedicated <a href= | |||
"/viral-vectors/lentivirus/">lentiviral plasmid page</a>.</td> | |||
</tr> | |||
<tr> | |||
<td>Retroviral</td> | |||
<td>Easy and safe to use</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/browse/article/102/">pBabe | |||
plasmids</a> - Mammalian retroviral gene | |||
expression, multiple selectable markers</li> | |||
<li><a href="http://www.addgene.org/8452/">pMKO.1 puro</a> - Mammalian | |||
retroviral shRNA expression</li> | |||
<li><a href="http://www.addgene.org/20672/">MSCV-IRES-GFP</a> - Mammalian | |||
retroviral gene expression with GFP selectable | |||
marker</li> | |||
<li><a href="http://www.addgene.org/31601/">LZRS-Rfa</a> or <a href= | |||
"/18656/">pMXs-gw</a> - Mammalian retroviral Gateway | |||
cloning vectors</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Adenoviral</td> | |||
<td>High transduction efficiency, but do NOT integrate into | |||
host genome. <a href="http://www.addgene.org/viral-vectors/aav/">View our AAV page for more information.</a></td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/16405/">pAdTrack-CMV</a> - Shuttle | |||
vector for transgene expression under a CMV | |||
promoter</li> | |||
<li><a href="http://www.addgene.org/16404/">pAdTrack</a> - Shuttle vector | |||
for transgene expression under your chosen | |||
promoter</li> | |||
<li><a href="http://www.addgene.org/16400/">pAdEasy®-1</a> - Recombine | |||
plasmids from the shuttle vectors into pAdEasy for | |||
viral production</li> | |||
<li><a href="http://www.addgene.org/16399/">AdEasier®-1 cells | |||
(strain)</a> - Bacterial strain that contains | |||
AdEasy®-1 plasmid</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
</tbody> | |||
</table><a href="#top">Back to Top</a> | |||
</section> | |||
<section id="special"> | |||
<h2>Reporters, shRNA expression, transgenics and genome | |||
modification</h2> | |||
<table class="empty-backbone-table table table-striped table-hover"> | |||
<thead> | |||
<tr> | |||
<th>Element</th> | |||
<th>Details</th> | |||
<th>Representative Empty | |||
Backbones</th> | |||
</tr> | |||
</thead> | |||
<tbody> | |||
<tr> | |||
<td>Promoter</td> | |||
<td>Measure promoter strength</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/16539/">pBV-Luc</a> - Luciferase | |||
reporter plasmid with very low basal activity</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>shRNA/RNAi</td> | |||
<td>For gene silencing experiments</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/10878/">pLKO.1 - TRC cloning | |||
vector</a> - Lentiviral plasmid for mammalian gene | |||
silencing</li> | |||
<li><a href="http://www.addgene.org/8452/">pMKO.1 puro</a> - Retroviral | |||
plasmid for mammalian gene silencing</li> | |||
<li><a href="http://www.addgene.org/12247/">pLVTHM</a> - lentiviral | |||
plasmid for mammalian gene silencing | |||
(tet-regulated)</li> | |||
<li><a href="http://www.addgene.org/1654/">L4440</a> - RNAi in C. | |||
elegans</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>Transgenic</td> | |||
<td>Expression in organisms</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/21270/">pBigT</a> - For making | |||
transgenic mice. Contains a loxP-flanked | |||
cassette</li> | |||
<li><a href="http://www.addgene.org/15037/">pBTG (pBigT-IRES-GFP)</a> - | |||
pBigT modified to contain IRES-GFP</li> | |||
<li><a href="http://www.addgene.org/browse/article/2323/">Hynes Lab | |||
Plasmids</a> - A system for Cre-regulated RNAi in | |||
vivo</li> | |||
<li><a href="http://www.addgene.org/browse/article/3655/">Dupuy Lab</a> | |||
and <a href="http://www.addgene.org/browse/article/5402/">Lehner Lab</a> | |||
vectors - Tools for nematode transgenesis</li> | |||
<li><a href="http://www.addgene.org/browse/article/4030/">Rosenberg Lab | |||
Plasmids</a> - Transgenic single-copy gene | |||
expression in E. coli</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
<tr> | |||
<td>TALENs</td> | |||
<td>Gene targeting</td> | |||
<td> | |||
<ul> | |||
<li><a href="http://www.addgene.org/TALEN/">TALEN kits</a> - Construct a | |||
custom TALEN array for genomic engineering</li> | |||
</ul> | |||
</td> | |||
</tr> | |||
</tbody> | |||
</table> | |||
</section> | |||
</html> |
Revision as of 14:16, 5 August 2015
Choosing A Plasmid Backbone
When choosing what plasmid backbone to use, you have many elements to consider. Here is a guide to Addgene's empty vector backbones. For the most part, we will assume that you want to express a gene; however, we have a section at the end for if you are studying a different genetic element or want to express shRNA.
<html> <section id="host">
Species-Specific Expression
If you want to drive expression of your favorite gene, you will need a plasmid with a promoter that will be functional in your host organism.
<thead> </thead> <tbody> </tbody>Host | Relevant Promoters | Representative Empty Backbones |
---|---|---|
Mammalian | CMV, SV40, EF1a, CAG |
|
Bacteria | Lac, T7, araBAD |
|
Yeast | GAL4, PGK, ADH1, ADE2, TRP1 |
|
Fly | UAS, MT |
|
Worm | unc-54, variety of worm gene promoters |
|
Insect / Baculovirus | Polyhedrin |
|
Xenopus | SP6 for transcription and injection |
|
<a href="#top">Back to Top</a>
</section>
<section id="tag">
Epitope Tag or Fusion Protein
Tags and fusion proteins are excellent tools for further understanding the function of your favorite gene. For example, fusing your protein to an epitope tag, such as Flag or HA, will allow you to easily identify your protein using an antibody against that epitope. This could allow you to conduct western blots or immunoprecipitations of your favorite gene even if you do not have an antibody against it. Another common scenario is fusing your protein to another protein, such as GFP, which allows you to visualize the cellular localization of your protein.
Just remember that when you are designing your plasmid you should keep your gene "in frame" with the fusion protein. This means that the final product should be translated as a single string of amino acids that preserves the sequence of your gene and of the fusion protein.
<thead> </thead> <tbody> </tbody>Tag or Fusion Protein | Common uses | Representative Empty Backbones |
---|---|---|
Flag | Epitope tag |
|
HA | Epitope Tag |
|
Myc | Epitope Tag |
|
6xHis | Epitope Tag |
|
GST | Protein Purification |
|
GFP | Localization |
|
Other Fluorescent Proteins | Localization | See full <a href="http://www.addgene.org/fluorescent-proteins/">Fluorescent Protein Guide</a> |
NLS | Nuclear Localization |
|
Myr | Membrane Localization |
|
For more information about tags, including amino acid sequences, see our <a href="http://www.addgene.org/mol_bio_reference/genetic_code/#tags">list of common tags</a>.
<a href="#top">Back to Top</a>
</section>
<section id="selection">
Selectable Markers
Regardless of your delivery method, it's unlikely that all of your cells will take up your plasmid. Thus, many plasmids have markers on them so that you can find or select for only the cells that received the plasmid.
<thead> </thead> <tbody> </tbody>Selectable Marker | Typical Host Organism | Representative Empty Backbones |
---|---|---|
Neomycin (G418) | Mammalian |
|
Puromycin | Mammalian |
|
Hygromycin | Mammalian |
|
Zeocin/Bleo | Mammalian |
|
GFP | Varies |
|
URA3 | Yeast |
|
TRP1 | Yeast |
|
LEU2 | Yeast |
|
HIS3 | Yeast |
|
<a href="#top">Back to Top</a>
</section>
<section id="viral">
Viral Expression and Packaging
Although transient expression is sufficient for some experiments, scientists often want to create stable cell lines in which the plasmid is incorporated into the host DNA. For mammalian cells, you can do this through viral delivery. <a href="http://www.addgene.org/viral-vectors/">Visit our viral vector page for more information.</a> Below are some common delivery methods:
<tbody> <thead> </thead> <tbody> </tbody>Delivery method | Advantages | Representative Empty Backbones |
---|---|---|
Lentiviral | Can transduce both dividing and nondividing cells | Addgene has an extensive collection of plasmids for
packaging and expression. See our dedicated <a href="/viral-vectors/lentivirus/">lentiviral plasmid page</a>. |
Retroviral | Easy and safe to use |
|
Adenoviral | High transduction efficiency, but do NOT integrate into host genome. <a href="http://www.addgene.org/viral-vectors/aav/">View our AAV page for more information.</a> |
|
<a href="#top">Back to Top</a>
</section>
<section id="special">
Reporters, shRNA expression, transgenics and genome modification
<thead> </thead> <tbody> </tbody>Element | Details | Representative Empty Backbones |
---|---|---|
Promoter | Measure promoter strength |
|
shRNA/RNAi | For gene silencing experiments |
|
Transgenic | Expression in organisms |
|
TALENs | Gene targeting |
|
</section> </html>