Difference between revisions of "Team:NAIT Edmonton/Protocols"

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       <center><h2>Ni-NTA Spin Kit</h2></center>
 
       <center><h2>Ni-NTA Spin Kit</h2></center>
 
<strong>NOTE:</strong> due to dissociation of urea used pH values of buffers will need to be check and if necessary adjusted <br>
 
<strong>NOTE:</strong> due to dissociation of urea used pH values of buffers will need to be check and if necessary adjusted <br>
<strong>NOTE:</strong> this protocol is suitable for use with frozen cell pellets. Cell pellets frozen for at least 30 minutes at -20°C can be lysed by re-suspending in lysis buffer and adding Benzonase Nuclease (3units/mL culture volume). Fresh pellets require sonication or homogenization in addition. To the addition of 3 units/mL culture volume Benzonase Nuclease and 1mg/mL culture volume lysozyme.<br>
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<strong>NOTE:</strong> this protocol is suitable for use with frozen cell pellets. Cell pellets frozen for at least 30 minutes at -20°C can be lysed by re-suspending in lysis buffer and adding Benzonase Nuclease (3units/mL culture volume). Fresh pellets require sonication or homogenization in addition. To the addition of 3 units/mL culture volume Benzonase Nuclease and 1mg/mL culture volume lysozyme.<br><br><br>
  
 
1. Thaw cells for 15 minutes and re-suspend in 700µL buffer B-7M urea and add 3 units/mL culture volume Benzonase Nuclease <br>
 
1. Thaw cells for 15 minutes and re-suspend in 700µL buffer B-7M urea and add 3 units/mL culture volume Benzonase Nuclease <br>
<strong>NOTE:</strong> Cells from 5 mL cultures are usually used, but culture volume used depends on protein expression level. Re-suspending pellet in 700µL buffer will allow recovery volume of cleared lysate of approx. 600µL<br>
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<strong>NOTE:</strong> Cells from 5 mL cultures are usually used, but culture volume used depends on protein expression level. Re-suspending pellet in 700µL buffer will allow recovery volume of cleared lysate of approx. 600µL<br><br>
 
2. Incubate cells with agitation for 15 minutes at room temperature. Solution should become translucent when lysis is complete. <br>
 
2. Incubate cells with agitation for 15 minutes at room temperature. Solution should become translucent when lysis is complete. <br>
<strong>NOTE:</strong> buffer B is the preferred lysis buffer, as the cell lysate can be analyzed directly by SDS-PAGE. If the cells or the protein do not solubilize buffer A must be used.<br>
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<strong>NOTE:</strong> buffer B is the preferred lysis buffer, as the cell lysate can be analyzed directly by SDS-PAGE. If the cells or the protein do not solubilize buffer A must be used.<br><br>
 
3. Centrifuge lysate at 12,000xg for 15-30 minutes at room temperature to pellet the cellular debris. Collect supernatant.<br>
 
3. Centrifuge lysate at 12,000xg for 15-30 minutes at room temperature to pellet the cellular debris. Collect supernatant.<br>
<strong>NOTE:</strong> save 20µL of the cleared lysate for SDS-PAGE analysis<br>
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<strong>NOTE:</strong> save 20µL of the cleared lysate for SDS-PAGE analysis<br><br>
 
4. Equilibrate a Ni-NTA spin column with 600µL buffer B-7M urea. Centrifuge for 2 minutes at 890xg (2900RPM).<br>
 
4. Equilibrate a Ni-NTA spin column with 600µL buffer B-7M urea. Centrifuge for 2 minutes at 890xg (2900RPM).<br>
 
<strong>NOTE:</strong> the spin columns should be centrifuged with an open lid to ensure that the centrifugation step is completed after 2 minutes.<br>
 
<strong>NOTE:</strong> the spin columns should be centrifuged with an open lid to ensure that the centrifugation step is completed after 2 minutes.<br>
5. Load up to 600µL of the cleared lysate supernatant containing the GxHis-tagged protein onto a pre-equilibrated Ni-NTA spin column. Centrifuge for 5 minutes at 270xg (1600RPM), and collect the flow-through<br>
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5. Load up to 600µL of the cleared lysate supernatant containing the GxHis-tagged protein onto a pre-equilibrated Ni-NTA spin column. Centrifuge for 5 minutes at 270xg (1600RPM), and collect the flow-through<br><br>
 
<strong>NOTE:</strong> to ensure sufficient binding, it is important not to exceed 270xg (1600RPM) when centrifuging Ni-NTA spin columns<br>
 
<strong>NOTE:</strong> to ensure sufficient binding, it is important not to exceed 270xg (1600RPM) when centrifuging Ni-NTA spin columns<br>
6. Wash Ni-NTA spin column with 600µL buffer C. Centrifuge for 2 minutes at 890xg (2900RPM)<br>
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6. Wash Ni-NTA spin column with 600µL buffer C. Centrifuge for 2 minutes at 890xg (2900RPM)<br><br>
 
7. Repeat step 6<br>
 
7. Repeat step 6<br>
 
8. Elute the protein with 200µL buffer E. Centrifuge for 2 minutes at 890xg (2900RPM) and collect the elute<br>
 
8. Elute the protein with 200µL buffer E. Centrifuge for 2 minutes at 890xg (2900RPM) and collect the elute<br>

Revision as of 02:42, 16 September 2015

Team NAIT 2015

Experimental Design

Go through our interactive experimental design flow chart! Many of our protocols are manufacturer specicfied; however, some are customized by us! Click on the flow chart boxes if the hand cursor appears to read more about our customized protocols.

PDFs of protocols can also be found

Theorizing our Sequences
Literature has shown certain proteins inherently stain in colour.
Looked up characteristics of said proteins
Isolated and identified the unique characteristics of said proteins so that we can manually write our own sequences and generate custom proteins.
Writing our Sequences
Went down to base pair level and wrote out our sequences with the defining characteristics identified in literature.
PCR
Digestion and Ligation
Transforming Bacteria
Validating the Transformation
Protein Isolation and Purification
SDS PAGE and Silver Staining