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Lab Floor Plan (with list of materials)
General microbiology protocols
Media Recipes
Reagent Recipes
Working with Antibiotics
Freezing -80 Stocks
Freezing Aliquots
Competition Assays
Generic PCR
Gradient PCR
Running DNA Gels
Protein Purification
Protein Sample Concentration
Cloning and gene manipulation
Commonly Used Plasmids
Plasmid Purification
Digest and Ligation
Gel Purification
Using the NEB #T1020 Monarch Kit
For a detailed protocol, or to download the full manual, visit www.neb.com/T1020
BEFORE YOU BEGIN:
- Add 4 volumes of ethanol (2 95%) to one volume of DNA Wash Buffer. (Needed to do the first time that the kit is used).
- All centrifugation steps should be carried out at 16,000 xg (-13,000 RPM).
- Please note: column holds 800 ul.
PROTOCOL STEPS:
- Run your digested DNA on an agarose gel. Use the 'wide combs' that will allow for more volume inside of them. Run the gel slowly (40-60 volts) for better band separation.
- Weigh empty micro-centrifuge tubes, and write the weight on the outside of the tube.
- Using the UV illuminator in Exx (across from Superlab), excise the DNA fragment from the agarose gel, taking care to trim excess agarose. Transfer to the 1.5 ml micro-centrifuge tube. Minimize exposure to UV light. Weigh the gel slice + micro-centrifuge tube and calculate the weight of just the agarose chunk. Agarose in a tube can be frozen at -20 and extracted later — this is a potential stopping point in the protocol.
- Turn on the small water bath and set to 50°C. Put in the DNA Elution Buffer bottle, ensuring that it does not tip over in the water bath.
- Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 ul buffer per 100 ul or 100 mg agarose).
- Incubate the sample at 50°C, vortexing periodically until the gel slice is completely dissolved (generally 5-10 minutes). For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 ul gel slice: 400 ul Gel Dissolving Buffer: 150ul water).
- Insert column into collection tube and load sample onto the column with the mixture. Remember that the column can hold only 800ul maximum; you may need to load 800ul; spin; discard flow-through; load remaining liquid; spin again. Spin for 1 minute, then discard flow-through.
- Re-insert column into collection tube. Add 200 ul DNA Wash Buffer and spin for 1 minute. Discarding flow-through.
- Repeat step 5 by adding 200 ul DNA Wash Buffer and spin for 1 minute.
- Transfer column to a clean 1.5 ml micro-centrifuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
- Add 6ul - 20ul of DNA Elution Buffer at 50°C to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA. Molecular grade water (pH 7-8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated.
Creating Competent E. coli Cells
Transformation
Gibson Assembly
Creating Lac- E. coli Mutants
Arabidopsis thaliana protocols
Creating Sterile Agar Plates
Sterile Seeding Protocol
Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments
Growth Stage Phenotype Definitions
Growth Conditions for ''Arabidopsis thaliana''
Measuring Light with HOBO Data Loggers
Inoculation of ''Arabidopsis thaliana'' with Microbes
Removal and DNA Extraction of Phyllosphere Microbes
ARISA
Measuring ''A. thaliana'' Phenotype using FIJI by Hand
DNeasy PowerSoil Protocol
Fiji Measurement
Making Boxes
Streptococcus pneumoniae protocols
Dual Layer Assays
Streptococcus DNA Extraction
Streptococcus Transformation
Streptococcus Growth Curve Protocol
Streptococcus Growth Curve and Cell Count in Liquid Media
Log Phase Growth Curve and Cell Count in Liquid Media
Streptococcus Bacteriocin (Dual Layer) Assays - Original
Streptococcus Bacteriocin (Dual Layer) Assays - Early Producer
Streptococcus Bacteriocin (Dual Layer) Assays - Light and Normal Target Lawns
Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria
Streptococcus suis protocols
Streptococcus suis Transformation
Measuring Absorbance in Streptococcus
Streptococcus DNA Extraction
Streptococcus Competence Induction
Peptide Synthesis
Peptide Cleavage
Mass Spectrometery
Plate Reader Assay and Growth Curve
Measuring Competence : Fixation and Flow Cytometry
Interactions Protocols
Zone of Inhibition Assay
Remote Molecular Biology
Effect of Laboratory Protocols on Student Learning
Interesting Podcats to Listen to When Doing Lab Work!
Cambridge protocols
Storage buffer
transformation of R5(2)-mCh-FL-BST and
expression
lysis and immobilization
Bio320 Microbe Species Wikipedia Pages
Getting started with MediaWiki
Consult the User's Guide for information on using the wiki software.