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=''In vivo''=
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== ''Escherichia coli'' ==
<div id="mainpage">
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[[Bacterial cell culture]]
==Contributing protocols==
===Add new protocols===
*[[Protocols/Create|'''Add a new lab- or person-specific protocol''']]
*[[Protocols/Consensus template|'''Add a consensus protocol''']] ([[Help:Consensus protocol|?]])


[[Chemically competent cells]]


[[Electrocompetent cells]]
===Improve protocols in development===
'''''Please Contribute!'''''
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:Needs attention]]-->
{{#dpl:category=Needs attention|category=Protocol}}


[[Electroporation]]
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[[Miniprep]]
==''In vivo''==


[[Colony PCR]]
{{protocols group|''Acetobacter xylinum''|2=
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[[Pulse-chase protein production]]
{{protocols group|''Arabidopsis thaliana''|2=
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}}


== Yeast ==
{{protocols group|''[[Escherichia coli]]''|2=
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{{#dpl:category=Escherichia coli|category=Protocol|titlematch=%:%}}


[[High Efficiency Transformation]]
}}


[[Yeast Colony PCR]]
{{protocols group|''Lactic Acid Bacteria''|2=
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:Lactobacillus]]-->
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[[Yeast DNA Prep]]
}}


[[Cell Cycle Arrest]]
{{protocols group|''[[Mesoplasma florum]]''|2=
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[[Fixing cells]]
}}


[[B-galactosidase assay]]
{{protocols group|Mouse|2=
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}}


[[Silver: Time-Lapse Microscopy|Time-Lapse Microscopy]]
{{protocols group|''Mycobacterium smegmatis''|2=
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:Mycobacterium smegmatis]]-->
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}}


[[Silver: Lysate for Western|Simple Yeast Lysate]] (for Western etc.)
{{protocols group|''[[Plasmodium falciparum]]''|2=
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}}


[[Assaying mating]]
{{protocols group|''[[Streptomyces:Information|Streptomyces]]''|2=
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}}


== T7 ==
{{protocols group|T7|2=
[[Studier Lysate Prep|Simple lysate protocol from plaque or lysate]]
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{{#dpl:category=T7|category=Protocol}}
}}


[[Cesium Chloride Purification of T7]]


== Mesoplasma florum ==
{{protocols group|[[Yeast]]|2=
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:Yeast]]-->
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}}


===Culture Media===


====ATCC 1161 Medium====
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<!-----------------------------------In vitro----------------------------------->
<!------------------------------------------------------------------------------->


Mix a base broth in a 1 liter 45mm cap bottle the following chemicals:
==''In vitro''==


{|
{{protocols group|Nucleic acids|2=
|-
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:In vitro]] and [[Category:DNA]] and [[Category:RNA-->
| Heart Infusion Broth (Difco 0038)
{{#dpl:category=DNA|category=RNA|category=In vitro|category=Protocol|nottitlematch=%:%}}
|    17.5 g
{{#dpl:category=DNA|category=RNA|category=In vitro|category=Protocol|titlematch=%:%}}
|-
}}
| Sucrose
|    40 g
|-
| Agar Noble (Difco 0142) if necessary
|   12 g
|-
| DI Water
|   710 ml
|-
|}


Autoclave at 121C for 20 minutes and cool to room temperture (broth) or 55C (Agar) on a 55C water bath.
{{protocols group|[[DNA]]|2=
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}}


Centrifuge 2 x 45 ml of Gibco 18180-059 liquid yeast extract at high speed to remove sediment. Pour only 45 ml into centrifuge bottles, eliminating as much as possible of the sediment in the bottom of the Gibco stock.
{{protocols group|[[RNA]]|2=
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}}


Thaw Sigma H-1138 heat inactivated horse serum.  For agar, bring both horse serum and centrifuged yeast extract to 37C.  Horse serum at 55C will sediment protein.
{{protocols group|[[Protein]]|2=
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}}


Assemble 150ml bottle top 0.22&mu; filter on the 1 liter bottle.  For agar, it is helpful to put the bottle on a foam insulating pad to delay cooling.  Add by vacuum filtration:
{{protocols group|[[Lipid]]|2=
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}}


{|
{{protocols group|Mammalian cell culture|2=
|-
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:In vitro]] and [[Category:Mammalian cell culture]]-->
| Horse serum (Sigma H1138)
{{#dpl:category=Mammalian cell culture|category=In vitro|category=Protocol}}
| 200 ml
}}
|-
| Yeast extract
| 90 ml
|-
| Penicillin G 500,000 U/ml
| 400 &mu;l
|-
| 2% phenol red
| 2 ml
|}
 
Adjust pH to 7.4 with 100 mM HCl.  Measured pH of as-made broth is 7.46, probably close enough.
 
Phenol red 2% solution must be neutralized with sufficient NaOH to convert it to the sodium salt.  The phenol red amount can be adjusted to control the depth of color to taste.
 
====SP-4 (ATCC 988) Medium====
 
Mix a base broth containing:


{|
{{protocols group|Plant specific protocols|2=
<!--To list a protocol here, "tag" the protocol page with the [[Category:Protocol]] and [[Category:In vitro]] and [[Category:Plant]]-->
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}}
|-
|-
| Mycoplasma broth base (BBL 111458)
| 3.5 g
|-
| Tryptone
| 10 g
|-
| Peptone
| 5.3 g
|-
| Glucose
| 5.0 g
|- 
| DI water
| 615 ml
|}
Adjust pH to 7.5 and autoclave at 121C for 20 minutes.
Centrifuge 2 x 17.5 ml of liquid yeast extract (Gibco 18180-059) at high speed to remove sediment.
Mix 2 g of Yeastolate (Difco 5577) in 100 ml DI water.
Sterile filter into the cooled medium:
{|
|-
| penicillin G 500,000 U/ml
| 400 &mu;l
|-
| 2% phenol red
| 2 ml
|-
| 2% Yeastolate (Difco 5577)
| 100 ml
|-
| Newborn calf serum
| 170 ml
|-
| liquid yeast extract
| 35 ml
|-
| 10x CMRL-1066 solution
| 50 ml
|}


=''In vitro''=
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<!------------------------------------------------------------------------------->
<!-----------------------------------In silico------------------------------------>
<!------------------------------------------------------------------------------->


==Nucleic acids==
==''In silico''==


[[Quantification of nucleic acids]]
{{protocols group|[[Cloning]]|2=
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[[Phenol/chloroform extraction]]
{{protocols group|[[Data analysis]]|2=
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}}


[[Nucleic acid precipitation]]
{{protocols group|[[Databases]]|2=
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}}


[[Denaturing acrylamide gel purification of nucleic acids]]
{{protocols group|[[Modelling]]|2=
=== DNA ===
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[[Agarose gel]]
{{protocols group|[[Sequence analysis]]|2=
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}}


[[Annealing Primers]]
{{protocols group|[[Structure analysis]]|2=
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}}


[[Designing primers]]
[[Tregwiki:Main_Page|Transcriptional Regulation]]


[[DNA Ligation]]


[[Library Generation]]
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<!------------------------------------------------------------------------------->
<!-----------------------------------OTHER--------------------------------------->
<!------------------------------------------------------------------------------->


[[PCR]]
==Other==


[[Phosphatase treatment of linearized vector]]
{{protocols group|General resources|2=
*[http://www.currentprotocols.com/WileyCDA/ Current Protocols] Most comprehensive source of protocols ranging from molecular biology to neuroscience.
*[http://mrw.interscience.wiley.com/emrw/9780471142720/home/ Current Protocols in Molecular Biology]
*[http://www.myjove.com/ JoVE] Journal of Visualized Experiments: An online research journal for publishing visualized (video-based) biological experiments.
*[http://protocol-online.org Protocol-online] Useful protocols and a popular discussion section.
*[http://www.springerprotocols.com Springer Protocols]  
}}


[[PNK Treatment of DNA Ends]]
{{protocols group|[[Aseptic Technique]]|2=
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[[Purification of DNA]]
{{protocols group|[[Flow cytometry]]|2=
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[[Reconstituting primers]]
{{protocols group|Microfluidics|2=
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[[Restriction digest]]
{{protocols group|[[Microscopy]]|2=
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[['Round-the-horn site-directed mutagenesis]]
{{protocols group|Plate reader|2=
*[[Endy:Victor3 plate reader|Plate reader usage protocols]]
}}


[[Sequencing DNA]]
{{protocols group|Miscellaneous|2=
<!--To list a protocol here, "tag" the protocol page with only [[Category:Protocol]]-->
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}}


[[Site-directed mutagenesis]]
{{protocols group|Need help with protocols?|2=
 
*[[Questions and Answers|Questions and Answers on OWW]]
[[Templiphi]]
}}
 
|}
=== RNA ===
{{show hide all}}
 
<sitesearch>
[[RNA Extraction]]
title=Search Protocols
 
qualifier=protocol
[[Northern Blot, 32P End-Labeled Probes |Northern Blot, <sup>32</sup>P End-Labeled Probes]]
target=
 
</sitesearch>
[[Probe Prep, 32P End-Labeled Probes|Probe Prep, <sup>32</sup>P End-Labeled Probes]]
</div>
 
__NOTOC__
[[RNase Protection Assay]]
 
[[In vitro transcription with T7 RNA polymerase]]
 
[[RNA Half-life]]
 
== Protein ==
 
[[Acrylamide Gels|Acrylamide gels and SDS-PAGE]]
 
[[Silver: Coomassie Stain|Coomassie Stain]]
 
[[Silver: Destain|Coomassie Destain]]
 
[[Sauer:Silver staining|Silver staining]]
 
[[Western Blot]]
 
= Microfluidics =
 
[[Design a Microfluidic Chip]]
 
[[Order a Microfluidic Chip from the Foundry]]
 
[[Operate Microfluidic Chemostat]]
 
= Microscopy =
 
[[Agarose Pads for Microscopy]]
 
[[Quantitative Microscopy]]
 
= Flow Cytometry =
 
[[Flow Calibration]]
 
= Miscellaneous =
 
[[Endy:Victor3 counteracting evaporation in long time courses|Counteracting evaporation from wells in a Victor3 plate reader]]
 
[[Pour plates|Pour LB plates]]
 
[[Pour YPD plates]]
 
[[Layered plates]]
 
[[S_N Chemostat Protocol]]
 
[[beta-Galactosidase Assay (A better Miller)]]
 
[[Silver: 10x PBS|10x PBS]]
 
[[Liquid Media]]

Revision as of 14:35, 30 July 2015

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Contributing protocols

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In vivo

Acetobacter xylinum

Arabidopsis thaliana

Escherichia coli

Lactic Acid Bacteria

Mesoplasma florum

Mouse

Mycobacterium smegmatis

Plasmodium falciparum

Streptomyces

T7


Yeast


In vitro

Nucleic acids

DNA

RNA

Protein

Lipid

Mammalian cell culture

Plant specific protocols

In silico

Cloning

Data analysis

Databases

Modelling

Sequence analysis

Structure analysis

Transcriptional Regulation


Other

General resources

Aseptic Technique

Flow cytometry

Microfluidics

Microscopy

Plate reader

Miscellaneous

Need help with protocols?

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