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stock is necessary | stock is necessary | ||
<p> If making glycerol stock, use 6mL LB broth so 800μL can be used for stock | <p> If making glycerol stock, use 6mL LB broth so 800μL can be used for stock | ||
+ | |||
+ | </p></td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table> | ||
+ | <center>Restriction Digestion<center> | ||
+ | <tr> | ||
+ | <th><h5>Introduction</h5></th> | ||
+ | <td><p>DNA concentration should be precise</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><h5>Materials</h5></th> | ||
+ | <td><p> 500ng DNA | ||
+ | <p> 1μL enzyme | ||
+ | <p> 5uL 10x cutsmart buffer | ||
+ | <p> 1X remainder milliQ | ||
+ | </p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th><h5>Procedure</h5></th> | ||
+ | <td><p>Protocol | ||
+ | <p> Leave for at least an hour in 37oC stationary incubator | ||
+ | <p> If using NEB BsaI-HF, add CIP (CALF intestinal phosphatase) and leave for another hour at 37oC | ||
</p></td></tr> | </p></td></tr> |
Revision as of 00:35, 31 October 2017
Home | Team | Collaborations | Project | Results |
Interlab Study | Safety | Human Practices | Attributions |
Interlab Study
We are very excited to announce we've transformed our DNA parts for the 2017 iGEM Interlab Study. Check out our Twitter page Follow @RPIiGEM for weekly updates on our project!
We will be using DNA parts from Plate 7 in the distribution kit and are looking forward to collecting our first round of data.
♦Process Flow♦
Steps with an asterisk are not required in the iGEM interlab study, rather they are control steps to preserve the DNA and ensure that if anything goes wrong, we have a stock to retransform We choose two colonies per plate so we can run biological duplicates. This can provide information on variability of compounds within a single organism (in this case, DH5a e. Coli) The iGEM plate reader protocol has a series of dilutions, so we will have to pay careful attention to those details when the time comes Form 1 can be filled out at any time, Form 2 requires spreadsheet data from plate reader, Form 3 is in regard to the culturing process |
♦Protocols♦
Introduction |
Used in conjunction with appropriate antibiotics to selectively plate |
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Materials |
LB Agar (NOT agarose), 35g/L appropriate antibiotic (ie, for 800mL LB Agar, add 800uL Amp, or 400Cm) |
Procedure |
Stir bar before autoclave If stir bar is forgotten, sterilize in EtOH to flame before dropping in autoclaved LB agar Mix appropriate amount of agar and DI water, dissolving completely to prevent burning agar Autoclave on liquid cycle (15 min is fine) Immediately after completing autoclave, cool on stir plate until not too hot to hold Add antibiotic Don't degrade antibiotic, but don't let cool too much and harden |
Introduction |
Use RuCl2 from -80 freezer |
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Materials |
100uL cells (thawed on ice) 250uL sterile SOC |
Procedure |
Process will take approximately 1h45min Add DNA to thawed cells on ice 30 minute incubation on ice for transformation 42oC heat shock for 45 seconds 1 minute on ice Add 250mL sterile SOC to 2mL tube Place in 37oC incubator at 250 RPM for 1 hour (recovery) |
Introduction |
Use this protocol for making permanent glycerol stocks, temporary glycerol stocks, and mini-prepping plasmid. |
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Materials |
5mL LB broth Antibiotic: 2.5 μL Chloramphenicol- Cam 50 (usually) |
Procedure |
Start 12-14 hours before commencing mini-prep Place in 37oC incubator at 250 RPM overnight CRISPR is a low-copy plasmid so 10-11mL overnights should be used, depending on whether or not glycerol stock is necessary If making glycerol stock, use 6mL LB broth so 800μL can be used for stock |
Introduction |
DNA concentration should be precise |
---|---|
Materials |
500ng DNA 1μL enzyme 5uL 10x cutsmart buffer 1X remainder milliQ |
Procedure |
Protocol Leave for at least an hour in 37oC stationary incubator If using NEB BsaI-HF, add CIP (CALF intestinal phosphatase) and leave for another hour at 37oC |