Difference between revisions of "Team:Kent/Experiments"
Line 36: | Line 36: | ||
<a href="#Overview">Overview</a> <br> | <a href="#Overview">Overview</a> <br> | ||
<a href="#Materials">Materials</a> <br> | <a href="#Materials">Materials</a> <br> | ||
+ | <li> Resuspended DNA </li> | ||
+ | <li> Competent cells </li> | ||
+ | <li> 2ml tube</li> | ||
+ | <li> 42˚C water bath</li> | ||
+ | <li> Petri dishes with LB agar and appropriate antibiotic</li> | ||
+ | <li> 37˚C incubator</li> | ||
+ | <li> 10pg/ul RFP Control</li> | ||
<a href="#Procedure">Procedure</a> <br> | <a href="#Procedure">Procedure</a> <br> | ||
+ | <ol> | ||
+ | <li>Thaw the competent cells on ice </li> | ||
+ | <li>Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control. </li> | ||
+ | <li>Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently.(<i>Make sure to keep the competent cells on ice. </i>) </li> | ||
+ | <li>Add 1 µL of the RFP Control to your control transformation. </li> | ||
+ | <li>Close tubes and incubate the cells on ice for 30 minutes. </li> | ||
+ | <li>Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. </li> | ||
+ | <li>Incubate the cells on ice for 5 minutes. </li> | ||
+ | <li>Add 200 μl of SOC media (<i>making sure that the broth does not contain antibiotics and is not contaminated</i>) to each transformation </li> | ||
+ | <li>Incubate the cells at 37˚C for 2 hours while the tubes are rotating or shaking. <b><i>2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin. </b></i> </li> | ||
+ | <li> </li> | ||
+ | <li> </li> | ||
+ | <li> </li> | ||
+ | <li> </li> | ||
+ | <li> </li> | ||
+ | <li> </li> | ||
+ | <li> </li> | ||
+ | </ol> | ||
<a href="#References">References</a> <br> | <a href="#References">References</a> <br> | ||
Revision as of 12:09, 30 June 2015
Experiments & Protocols
Contents
Competent Cells
Transformation Protocol
Miniprep
Competent Cells
Overview- Overnight culture of VS45 cells are back-diluted to OD600 0.1 in 250 ml LB broth
- The cells are then grown at 37˚C to OD600 0.6 and then harvested by centrifugation.
- The cells are resuspended in 100 ml of prechilled buffer and incubated on ice for 60 minutes.
- Harvest again by centrifugation (at 4˚C), and resuspended in 5 ml of pre-chilled buffer.
- The resuspended cells can then be aliquoted (on ice), frozen using dry ice or liquid nitrogen, and stored at -80˚C.
Transformation Protocol
OverviewMaterials
- Thaw the competent cells on ice
- Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
- Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently.(Make sure to keep the competent cells on ice. )
- Add 1 µL of the RFP Control to your control transformation.
- Close tubes and incubate the cells on ice for 30 minutes.
- Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
- Incubate the cells on ice for 5 minutes.
- Add 200 μl of SOC media (making sure that the broth does not contain antibiotics and is not contaminated) to each transformation
- Incubate the cells at 37˚C for 2 hours while the tubes are rotating or shaking. 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
Miniprep
Overview
Procedure
- Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer it to a microcentrifuge tube.
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
- Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through.
- Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
- Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through
- Centrifuge for 1 min to remove residual wash buffer.
- Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
- Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.