Team:Harvard/GenProtocols
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+ | <html><a href = "https://2008.igem.org/Team:Harvard"><img src="https://static.igem.org/mediawiki/2008/b/b9/Harvard_logo.png"></a></html> | ||
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+ | {{Template:Main}} | ||
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+ | {| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|} | ||
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+ | {|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%" | ||
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+ | =General Protocols= | ||
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+ | __TOC__ | ||
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+ | ==Transforming <i>Shewanella oneidensis</i>== | ||
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+ | #Grow bacteria to log phase (OD 0.4-0.6) | ||
+ | #Aliquot 1mL of culture to a 1.5mL tube. Make as many aliquots as you need for the number of plasmids you need to transform | ||
+ | #Centrifuge aliquots at 12,000g for 1 minute | ||
+ | #Remove media and wash with 0.33 volumes (330μL) of 1M sorbitol | ||
+ | #Centrifuge at 12,000g for 1 minute | ||
+ | #Remove wash and resuspend in 0.05 volumes (40μL) 1M sorbitol. | ||
+ | #Place tubes in ice and use within 15 minutes. | ||
+ | #Add 100-500ng of plasmid DNA to the tube | ||
+ | #Electroporate at 0.55kV | ||
+ | #Flush cuvette gently with 800ul SOC | ||
+ | #Allow to recover for 1-2 hrs at 30 degrees C, shake at 200rpm | ||
+ | #Spin down cells briefly, pour off most of supernatant, gently resuspend cells and plate | ||
+ | #Colonies will appear overnight at 30 degrees C, although they will be significantly smaller than E. coli grown for a similar period of time. After 2 nights of growth, colonies become visibly pinkish orange. | ||
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+ | Freezing these cells for later use is not recommended. Even with storage at -80C, subsequent transformations often fail. | ||
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+ | Efficiency is variable; 200ng DNA general yields 20-100 colonies. | ||
==Ligation protocol, using Roche Rapid Ligation Kit== | ==Ligation protocol, using Roche Rapid Ligation Kit== | ||
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'''Reagents''' | '''Reagents''' | ||
- | Make sure buffers are completely thawed before use. The 2x rapid ligation buffer contains a reducing agent that looks like white flakes when thawed. These white flakes must be completely dissolved back into the buffer before use | + | Make sure buffers are completely thawed before use. The 2x rapid ligation buffer contains a reducing agent that looks like white flakes when thawed. These white flakes must be completely dissolved back into the buffer before use. |
- | ''' | + | '''Troubleshooting''' |
- | + | What to try if your ligation isn’t working (in this order). | |
+ | # Make sure your gel box uses long wave UV. Short wave UV will cause thymine dimer formation that can destroy your sticky ends (e.g. the TTAA overhang of EcoRI) | ||
+ | # Repeat the ligation. Try a range of insert to vector ratios from 2:1 to 10:1. | ||
+ | # Review all of your digestion steps – are you sure you cut the vector and the insert with the correct enzymes? Did you let it digest for long enough? | ||
+ | # Try a different tube of enzymes kit. | ||
+ | # Still not working? Send the parts you are trying to clone for sequencing. You may not be cloning what you think you are cloning. | ||
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==Bacterial Transformation Protocol== | ==Bacterial Transformation Protocol== | ||
- | ===Chemically competent | + | ===Chemically competent E. coli=== |
====To make chemically competent cells==== | ====To make chemically competent cells==== | ||
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==Restriction Digest, using [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp New England Biolabs] (NEB) Enzymes== | ==Restriction Digest, using [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp New England Biolabs] (NEB) Enzymes== | ||
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Extension: 15-30s/kb @ 72°C (20s is usually enough for plasmids) | Extension: 15-30s/kb @ 72°C (20s is usually enough for plasmids) | ||
- | ==Colorimetric | + | ==Colorimetric LBB Assay Protocol (from Colleen Hansel)== |
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LBB Reagent: 0.04% LBB in 45 mM acetic acid | LBB Reagent: 0.04% LBB in 45 mM acetic acid | ||
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Note: The linear range for the LBB colorimetric method is 10-50 uM. If your samples are above this limit, dilute accordingly to bring into linear range. Do not force standard curve through zero – there is a background for the LBB solution. | Note: The linear range for the LBB colorimetric method is 10-50 uM. If your samples are above this limit, dilute accordingly to bring into linear range. Do not force standard curve through zero – there is a background for the LBB solution. | ||
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==[http://www.genewiz.com/preparesample.aspx Setting up Samples for Sequencing]== | ==[http://www.genewiz.com/preparesample.aspx Setting up Samples for Sequencing]== | ||
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- | ==Testing Inducible | + | ==Testing IPTG Inducible GFP Systems== |
#Grow overnight culture of cells (including positive control w/o repressor and negative control w/o fluorescence) | #Grow overnight culture of cells (including positive control w/o repressor and negative control w/o fluorescence) | ||
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# Use the vacuum centrifuge (on widget group's bench) to evaporate the remaining ethanol | # Use the vacuum centrifuge (on widget group's bench) to evaporate the remaining ethanol | ||
# Resuspend in 10-15μL sterile nuclease-free water. Be sure to pipet the water repeatedly onto the sides of the bottom of the tube and vortex gently a few times. Spin down everything. | # Resuspend in 10-15μL sterile nuclease-free water. Be sure to pipet the water repeatedly onto the sides of the bottom of the tube and vortex gently a few times. Spin down everything. | ||
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Latest revision as of 05:38, 29 October 2008
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