Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/23 July 2015"
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The lysate seems quite viscous, and seems to have some stringy, thick and gluttonous strands suspended in the middle. Even when not observed under blue light, the lysate solution appears an obvious light green color. We hypothesize that this may be due to the super-folded GFP being super concentrated in the lysate. Also, when lysate, diluted in 1X binding buffer, is added to the resin and the first wash buffer is added (20mM imidazole), the resulting solutions appears white, and a bit foamy or soapy. This contrasts with our previous experience with IMAC Batch purification, when the solution appeared mostly clear. We also attribute this to our lysate being super concentrated, especially when compared to our previous batch of Tamura, which was suspended in excessive amounts of lysis buffer. Despite these unexpected observations, we proceeded with the protocol, and collected Fractions 1-3. When the final elution buffer was added (250mM imidazole), the resin mixed with lysate was transferred into a column. Two layers appeared in the column, a viscous white one on bottom and a clearer one on top. The fraction collects at a slow pace, one drop every 15-20 seconds. Also, it's interesting that even though the strip buffer hasn't been added, the resin already seems to have been stripped. It is no longer blue. | The lysate seems quite viscous, and seems to have some stringy, thick and gluttonous strands suspended in the middle. Even when not observed under blue light, the lysate solution appears an obvious light green color. We hypothesize that this may be due to the super-folded GFP being super concentrated in the lysate. Also, when lysate, diluted in 1X binding buffer, is added to the resin and the first wash buffer is added (20mM imidazole), the resulting solutions appears white, and a bit foamy or soapy. This contrasts with our previous experience with IMAC Batch purification, when the solution appeared mostly clear. We also attribute this to our lysate being super concentrated, especially when compared to our previous batch of Tamura, which was suspended in excessive amounts of lysis buffer. Despite these unexpected observations, we proceeded with the protocol, and collected Fractions 1-3. When the final elution buffer was added (250mM imidazole), the resin mixed with lysate was transferred into a column. Two layers appeared in the column, a viscous white one on bottom and a clearer one on top. The fraction collects at a slow pace, one drop every 15-20 seconds. Also, it's interesting that even though the strip buffer hasn't been added, the resin already seems to have been stripped. It is no longer blue. | ||
| + | |||
| + | '''Transformation"" | ||
| + | |||
| + | Fasih continues to prepare a control to which we can compare our co-spin of wild-type silk and recombinant silk with GFP. The control is essentially pure GFP. A transformation is done using iGEM registry parts and competent cells. | ||
| + | |||
| + | '''Start-Up Culture''' | ||
| + | Vinson and Olivia's 9-mers sequences have been cloned into cells and plated! In a culture tube, 10mL of LB and 10ul of chloramphenicol is prepared. (The cells have psb1c3 as the vector and therefore chloramphenicol instead of ampicillin is used). A colony is picked with a pipette tip and swirled into the culture tube, which is placed into a shaking incubator at 37 degrees Celsius and left overnight. | ||
Revision as of 21:24, 24 July 2015
2mL of fresh resin was added to the a pre-chilled 50mL Falcon tube. The contents were centrifuged to discard the ethanol supernatant. The standard IMAC Batch Purification protocol was followed, as in previous purification trials.
Observations
The lysate seems quite viscous, and seems to have some stringy, thick and gluttonous strands suspended in the middle. Even when not observed under blue light, the lysate solution appears an obvious light green color. We hypothesize that this may be due to the super-folded GFP being super concentrated in the lysate. Also, when lysate, diluted in 1X binding buffer, is added to the resin and the first wash buffer is added (20mM imidazole), the resulting solutions appears white, and a bit foamy or soapy. This contrasts with our previous experience with IMAC Batch purification, when the solution appeared mostly clear. We also attribute this to our lysate being super concentrated, especially when compared to our previous batch of Tamura, which was suspended in excessive amounts of lysis buffer. Despite these unexpected observations, we proceeded with the protocol, and collected Fractions 1-3. When the final elution buffer was added (250mM imidazole), the resin mixed with lysate was transferred into a column. Two layers appeared in the column, a viscous white one on bottom and a clearer one on top. The fraction collects at a slow pace, one drop every 15-20 seconds. Also, it's interesting that even though the strip buffer hasn't been added, the resin already seems to have been stripped. It is no longer blue.
Transformation""
Fasih continues to prepare a control to which we can compare our co-spin of wild-type silk and recombinant silk with GFP. The control is essentially pure GFP. A transformation is done using iGEM registry parts and competent cells.
Start-Up Culture Vinson and Olivia's 9-mers sequences have been cloned into cells and plated! In a culture tube, 10mL of LB and 10ul of chloramphenicol is prepared. (The cells have psb1c3 as the vector and therefore chloramphenicol instead of ampicillin is used). A colony is picked with a pipette tip and swirled into the culture tube, which is placed into a shaking incubator at 37 degrees Celsius and left overnight.