Difference between revisions of "MWSU protocols"

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(Golden Gate Assembly Protocol for BsmB1)
 
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'''Purification of DNA'''
 
'''Purification of DNA'''
Golden Gate Assembly Protocol for BsmB1
 
  
GGA mixture contains:
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[[Isolation of Genomic DNA from Bacteria]]
  
1 µL (50 ng) Plasmid
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[[Gel Purification New England BioLabs Monarch]]
1 µL Promoter
 
1 µL 10X Promega Ligase Buffer
 
6 µL dH2O
 
0.5 µL BsmB1 high fidelity (HF) restriction enzyme
 
9.5 µL volume
 
  
Place tubes into 55°C water bath for 15 minutes.
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'''PCR'''
Add 0.5 µL T4 DNA Ligase to the mixture.
 
  
Turn on the PCR machine. Put tube into machine.
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[[iPCR]]
Program it for the following cycles:
 
• 20 cycles
 
• 55°C for 10 minutes
 
• 37°C for 1 minute
 
• 16°C for 1 minute
 
• 22°C holding temperature
 
  
This DNA ligation is ready for transformation.
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[[Gradient/Standard PCR]]
You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
 
  
Transformation of GGA BsmB1
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[[Resuspending Oligos]]
  
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product.  Place the entire content of the tube on the plate, spread and incubate.
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[[Davidson_Missouri_W/colony_PCR | Colony PCR]]
  
 +
[[Template Preparation for RT-qPCR]]
  
[[Isolation of Genomic DNA from Bacteria]]
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[[New Chaperone PCR]]
 +
 
 +
[[LongAmp PCR]]
  
'''PCR'''
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'''Recombinant DNA Production'''
  
[[Standard PCR]]
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[[Zymo Research Plasmid Minipreps]]
  
[[Resuspending Oligos]]
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[[Zymo Research Clean and Concentrate for Plasmid DNA]]
  
[[Davidson_Missouri_W/colony_PCR | Colony PCR]]
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[[Golden Gate Assembly Protocol]]
  
'''Recombinant DNA Production'''
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[[Golden Gate Assembly Single Molecule Protocol]]
  
 
[[Pouring an Agarose Gel]]
 
[[Pouring an Agarose Gel]]
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[[Reducing Background from Double Digested Vector]]
 
[[Reducing Background from Double Digested Vector]]
 +
 +
[[File: PClone_Procedure_for_GCAT_SB_Workshop_2014_new_version.pptx]]
  
 
'''Ligation and Transformation'''
 
'''Ligation and Transformation'''
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[[Ligation and Transformation]]
 
[[Ligation and Transformation]]
  
[[Bacterial Media]]
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[[Electroporation Transformation]]
 +
 
 +
[[SOC Protocol for Transformations]]
  
 
'''Screening Clones'''
 
'''Screening Clones'''
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[[DNA Sequencing at Iowa State University]]
 
[[DNA Sequencing at Iowa State University]]
 +
 +
[[DNA Sequencing at Eurofins]]
  
 
[[What to do with a new clone]]
 
[[What to do with a new clone]]
 +
 +
[[How to make a new Registry page]]
  
 
'''Measuring Phenotypes'''
 
'''Measuring Phenotypes'''
  
 
[[Measuring Fluorescence in Bacteria]]
 
[[Measuring Fluorescence in Bacteria]]
 +
 +
[[Camera Settings for Taking Pictures of Plates]]
 +
 +
'''DNA and E coli'''
 +
 +
[[GCAT Library of Quality Parts]]
 +
 +
[[MWSU Freezer Parts]]
 +
 +
'''Working With Bacteria'''
 +
 +
[[Bacterial Media]]
 +
 +
[[Washing Beads]]
 +
 +
[[Cleaning Plate Replication Pads]]

Latest revision as of 20:08, 14 June 2018

Purification of DNA

Isolation of Genomic DNA from Bacteria

Gel Purification New England BioLabs Monarch

PCR

iPCR

Gradient/Standard PCR

Resuspending Oligos

Colony PCR

Template Preparation for RT-qPCR

New Chaperone PCR

LongAmp PCR

Recombinant DNA Production

Zymo Research Plasmid Minipreps

Zymo Research Clean and Concentrate for Plasmid DNA

Golden Gate Assembly Protocol

Golden Gate Assembly Single Molecule Protocol

Pouring an Agarose Gel

BioBrick Digestions for Fragment and Vector Preparation

Fragment Purification

Gibson Assembly

Direct Synthesis with Overlapping Oligos

Annealing Oligos for Cloning

Ethanol Precipitation of Vector DNA

Reducing Background from Double Digested Vector

File:PClone Procedure for GCAT SB Workshop 2014 new version.pptx

Ligation and Transformation

BioBrick Ligations

Ligation and Transformation

Electroporation Transformation

SOC Protocol for Transformations

Screening Clones

Diagnostic RP Digestion for Checking Insert Size

DNA Sequencing at Iowa State University

DNA Sequencing at Eurofins

What to do with a new clone

How to make a new Registry page

Measuring Phenotypes

Measuring Fluorescence in Bacteria

Camera Settings for Taking Pictures of Plates

DNA and E coli

GCAT Library of Quality Parts

MWSU Freezer Parts

Working With Bacteria

Bacterial Media

Washing Beads

Cleaning Plate Replication Pads