Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/7 July 2015"

Revision as of 00:43, 10 July 2015

Lysis of Cells, Testing the Protocol with Fractions 1-5

Observations: Purified Tamura still slightly cloudy and not completely transparent solution. The white cell debris pellets at bottom still contain specks of green and fluoresce when observed under UV light, indicating incomplete lysis and not all protein is extracted. This is likely because Tamura is suspended in very dilute lysis buffer, making it more difficult for reagents to react in a homogenous manner, especially since such small volumes and amounts of reagents are used. To troubleshoot, lysis protocol is repeated with remaining cell pellet from final centrifugation step.

Lysis Protocol

1. Add Lysozyme.

2. Incubate on ice for 30 minutes.

3. Add PMSF solution, and while swirling, slowly add Deoxycholic Acid in powder form.

4. Incubate at 37 degrees Celsius for 20 minutes.

5. Add DNAse.

6. Incubate on rocking incubator at room temperature for 30 minutes.

7. Centrifuge sample for 15 minutes at 3,000g at 4 degrees Celsius.

8. Collect supernatant and store at -20 degrees Celsius.

@@ Line 288: / Line 288: @@
 <u>Lysis of Cells, Testing the Protocol with Fractions 1-5</u>
+Observations: Purified Tamura still slightly cloudy and not completely transparent solution. The white cell debris pellets at bottom still contain specks of green and fluoresce when observed under UV light, indicating incomplete lysis and not all protein is extracted. This is likely because Tamura is suspended in very dilute lysis buffer, making it more difficult for reagents to react in a homogenous manner, especially since such small volumes and amounts of reagents are used. To troubleshoot, lysis protocol is repeated with remaining cell pellet from final centrifugation step.
 '''Lysis Protocol'''