Lidstrom:Protocols

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(E. Coli)
(DNA)
(42 intermediate revisions not shown.)
Line 4: Line 4:
*[[Lidstrom:Health & Safety|Health & Safety]]
*[[Lidstrom:Health & Safety|Health & Safety]]
*[[Lidstrom:Finding Chemicals|Finding Chemicals]]
*[[Lidstrom:Finding Chemicals|Finding Chemicals]]
 +
*[[Lidstrom:Dishwasher |Dishwasher]]
*[[Lidstrom:Autoclave |Autoclave]]
*[[Lidstrom:Autoclave |Autoclave]]
*[[Lidstrom:Sterile Technique | Sterile technique]]
*[[Lidstrom:Sterile Technique | Sterile technique]]
Line 12: Line 13:
==Cell Culture:==
==Cell Culture:==
*[[Lidstrom:Tube Spec|Tube Spec]]
*[[Lidstrom:Tube Spec|Tube Spec]]
 +
*[[Lidstrom:M9|M9 recipe]]
 +
*[[Lidstrom:TB|Terrific Broth recipe]]
 +
*[[Lidstrom: Bioscreen|Bioscreen Setup]]
 +
*[[Lidstrom: EZ amino acid stock recipe|EZ amino acid stock recipe]]
==DNA/RNA:==
==DNA/RNA:==
===DNA===
===DNA===
 +
====Isolation ====
 +
*[[Lidstrom:QIAquick Gel Extraction Kit Protocol|QIAquick Gel Extraction Kit Protocol]]
 +
*[[Lidstrom:Genomic DNA extraction|Genomic DNA minikit extraction]]
 +
*[[Lidstrom:Genomic DNA extraction|Genomic DNA phenol/chloroform extraction]]
 +
 +
====Manipulation====
*[[Lidstrom:Building with DNA|Intro: Building with DNA (in progress)]]
*[[Lidstrom:Building with DNA|Intro: Building with DNA (in progress)]]
-
*[[Lidstrom:Colony PCR| Colony PCR]]
+
*[[Lidstrom:Overlap Extension PCR| Overlap Extension PCR (in progress)]]
 +
*[[Qiagen_Buffers| Qiagen Buffer Recipes]]
 +
 
 +
=====Fancy Manipulation=====
 +
*[[Lidstrom:Site-Directed Mutagenesis|Site-Directed Mutagenesis (in progress)]]
 +
*[[Lidstrom:λ-Red Mediated Gene Knockouts|λ-Red Mediated Gene Knockouts (in progress)]]
 +
*[[Dorman:P1_phage_%28lysogenic%29|Phage Lysate Prep]]
 +
*[[Lidstrom:P1 Phage Transduction|P1 Phage Transduction (in progress)]]
 +
 
 +
==== Restriction Cloning ====
*[[Lidstrom:Digestion with Restriction Enzymes|Digestion with Restriction Enzymes]]
*[[Lidstrom:Digestion with Restriction Enzymes|Digestion with Restriction Enzymes]]
-
*[[Lidstrom:Diluting Primers | Diluting Primers]]
 
-
*[[Lidstrom:Gibson Assembly| Gibson Assembly (In progress)]]
 
*[[Lidstrom:Ligation |Ligation (in progress)]]
*[[Lidstrom:Ligation |Ligation (in progress)]]
-
*[[Lidstrom:Loading Dye Recipe|Loading Dye Recipe]]
+
 
-
*[[Lidstrom:Miniprep |Miniprep (in progress)]]
+
==== Gibson Cloning ====
-
*[[Lidstrom:Oligo Orders | Oligo Orders (in progress)]]
+
*[[Lidstrom:Gibson Assembly| Gibson Assembly (In progress)]]
-
*[[Lidstrom:Overlap Extension PCR| Overlap Extension PCR (in progress)]]
+
 
 +
====Obervation====
 +
*[[Lidstrom:Colony PCR| Colony PCR]]
 +
*[[Lidstrom: Agarose Gel Electrophoresis|Agarose Gel Electrophoresis]]
 +
*[[Lidstrom:Sequencing with GeneWiz|Sequencing with GeneWiz]]
 +
 
 +
====Misc.====
*[[Lidstrom:PCR| PCR (in progress)]]
*[[Lidstrom:PCR| PCR (in progress)]]
 +
*[[Lidstrom:Loading Dye Recipe|Loading Dye Recipe]]
*[[Lidstrom:Purifying DNA|Purifying DNA (in progress)]]
*[[Lidstrom:Purifying DNA|Purifying DNA (in progress)]]
-
*[[Lidstrom:Sequencing with GeneWiz|Sequencing with GeneWiz]]
+
*[[Lidstrom:Miniprep |Miniprep (in progress)]]
-
*[[Lidstrom:Site-Directed Mutagenesis|Site-Directed Mutagenesis (in progress)]]
+
*[[Lidstrom:Oligo Orders | Oligo Orders]]
-
*[[Lidstrom:Transformation| Transformation (in progress)]]
+
*[[Lidstrom:Diluting Primers | Diluting Primers]]
-
===RNA===
+
==Protein==
 +
*[[Lidstrom: SDS-PAGE | SDS-PAGE Protein Gels]]
==E. Coli==
==E. Coli==
 +
*[[Lidstrom:E. Coli Vector Compatibility| E. Coli Vector Compatibility  (In Progress)]]
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]]
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]]
 +
*[[Lidstrom:Chemical Transformation|Chemical Transformation]]
 +
*[[Lidstrom:Electroporation|Electroporation]]
 +
*[[Lidstrom:Competent Cells|Competent Cell Lines]]
 +
 +
==Enzyme Assays==
 +
 +
===General===
 +
*[[Lidstrom: Plate Reader| Plate Reader Setup]]
 +
 +
===in vitro pathway assays (CO<sub>2</sub> project)===
 +
*[[Lidstrom:in vitro Pathway Assay: Cell Prep| Cell Prep (in progress)]]
 +
*[[Lidstrom:in vitro Pathway Assay: Crude Extract Prep| Crude Extract Prep (in progress)]]
 +
*[[Lidstrom:in vitro Pathway Assay: Sample Analysis via UPLC-MS/MS | Sample Analysis via UPLC-MS/MS (in progress)]]
-
==Electrocompetent Cells==
+
== Metabolomics ==
-
*You can make your own electro-competent cells for electroporation.
+
=== CO2 project ===
-
**From Amada's past mentor in undergrad:
+
*[[Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project|Measuring 13C Incorporation Into Protein - CO2 Project]]
-
***Grow the cells overnight
+
** [[Lidstrom:13C Incorporation Into Protein - Data Analysis]]
-
***Inoculate from this the next morning: generally use 200 uL/50 mL
+
-
***Let grow for about 3-4 h (OD 0.4-0.6 or so),  then centrifuge and wash 3 times with 10% glycerol (everything on ice).
+
-
***Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
+
-
****Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care.  See [[Electrocompetent_Cells| this page]].  Would allow you to calculate efficiency.
+

Revision as of 09:07, 31 March 2013

Back home: Lidstrom

Contents

General Lab:

Cell Culture:

DNA/RNA:

DNA

Isolation

Manipulation

Fancy Manipulation

Restriction Cloning

Gibson Cloning

Obervation

Misc.

Protein

E. Coli

Enzyme Assays

General

in vitro pathway assays (CO2 project)

Metabolomics

CO2 project

Personal tools