Difference between revisions of "Team:Pasteur Paris/Protocols/CellBio"
| Line 56: | Line 56: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>Bacteria transformation</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 68: | Line 68: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>Liquid culture</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 80: | Line 80: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>Bacterial stocks</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 92: | Line 92: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>DNA extraction from bacterial culture</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 104: | Line 104: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>Enzymatic digestion</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 117: | Line 117: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>Electrophoresis on agar gel</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 130: | Line 130: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>DNA Gel extraction</p> |
</div> | </div> | ||
</div> | </div> | ||
| Line 142: | Line 142: | ||
<div class="vignette_text"> | <div class="vignette_text"> | ||
| − | <p> | + | <p>Ligation of plasmid with DNA insert</p> |
</div> | </div> | ||
</div> | </div> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| Line 393: | Line 383: | ||
act_vign.style.order="16"; | act_vign.style.order="16"; | ||
act_pan.style.order="19"; | act_pan.style.order="19"; | ||
| − | |||
| − | |||
| − | |||
| − | |||
break; | break; | ||
} | } | ||
| Line 438: | Line 424: | ||
act_vign.style.order="16"; | act_vign.style.order="16"; | ||
act_pan.style.order="17"; | act_pan.style.order="17"; | ||
| − | break; | + | break; |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
} | } | ||
} | } | ||
| Line 483: | Line 465: | ||
act_vign.style.order="16"; | act_vign.style.order="16"; | ||
act_pan.style.order="18"; | act_pan.style.order="18"; | ||
| − | break; | + | break; |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
} | } | ||
} | } | ||
Revision as of 16:15, 13 September 2018
Molecular Biology: general protocols
Here we present the basic molecular biology methods we used throughout the project to amplify our plasmids, linearize them and insert our sequences, retrieve them from bacteria and express proteins.
Bacteria transformation
Liquid culture
Bacterial stocks
DNA extraction from bacterial culture
Enzymatic digestion
Electrophoresis on agar gel
DNA Gel extraction
Ligation of plasmid with DNA insert
Aim
Insert a plasmid of interest into competent bacterial cells, in order to replicate them.
Aim
Grow a colony that have successfully been transformed with one or several plasmids in order to replicate plasmid or to express a protein.
Aim
Stock bacterial culture at -80 °C.
Materials
- Desired bacterial cultures on petri dish
- Sterile LB media
- chloramphenicol (25mg/mL) or carbenicilline(50mg/mL)
- glycerol 50%
Procedure
In advance:
- Prepare a stock solution of LB + desired antibiotic in 50 mL falcon tube depending on how many culture you would like to stock in glycerol.
- Prepare a sterile stock solution of glycerol 50 %.
- In 15 ml sterile falcon, add 5 mL of LB media
- Vortex the stock solution of antibiotic and add 5 µL to the LB
- Using an inoculation loop, touch gently a colony of transformed bacteria from the petri dish, plastic side facing you.
- Immerse and dip the inoculation loop in the liquid media and stir.
- Place the liquid culture in the incubator at 37˚C 180 rpm for 16h.
- After 16 hours, centrifuge the tubes 5 minutes at 3000 rpm.
- Discard supernatant.
- Resuspend the pellet in 5mL of LB.
- Discard supernatant.
- Resuspend the pellet in 1 mL of LB + antibiotic.
- In a 125 ml erlenmeyer, add 1 mL of bacterial culture in 24 mL of LB + antibiotic.
- Incubate the culture at 37˚C 180 rpm.
- Measure the OD every hour for the first 3 hours and then every 20 minutes.
- When 0,6 < OD < 0,7, withdraw 5 mL of the bacterial liquid culture and add 5 mL of glycerol 50 %.
- Vortex.
- Aliquot the 10 mL into sterile cryotubes.
- Place into dry ice and freeze at -80˚C.
Aim
Retrieve amplified plasmids from a liquid culture of transformed bacteria. According to the liquid culture volume, we used the QIAfilter Plasmid Purification kit (for 25 mL culture) or the QIAprep Spin Miniprep kit (for 5 mL culture) from Qiagen.
Aim
Perform restriction enzyme digestion in order to recover linear backbones of the plasmids, extract our inserts from commercial plasmid, or check the success of a ligation.
Aim
Separate DNA fragments according to their molecular weight after an enzymatic digestion, in order to purify inserts or to analyse a plasmid.
Aim
: Extract DNA from an agar gel after an electrophoresis. We used the QIAquick Gel Extraction Kit provided by Qiagen.
Aim
Insert a DNA fragment with appropriate overlaps into a linearized plasmid. We used the In-Fusion HD Cloning Plus provided by Ozyme.