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Team:Nevada/Week 15
From 2012.igem.org
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[[Team:Nevada/Week 18| Week 18]] | | [[Team:Nevada/Week 18| Week 18]] | | ||
[[Team:Nevada/Week 19| Week 19]] | | [[Team:Nevada/Week 19| Week 19]] | | ||
| - | [[Team:Nevada/ | + | [[Team:Nevada/Final Weeks| Final Weeks]] | |
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==August 27== | ==August 27== | ||
| + | :Joe: fusion and transformation | ||
| + | |||
| + | :Michelle: | ||
| + | :::Pellet large scale expression to prepare for purification of M2-BL21 glycerol stock | ||
| + | :::+ LB AMP-CM. | ||
| + | |||
| + | LJustin and Dafne: | ||
| + | :::Determine optimal Imidazole concentration to utilize in Ni-column purification | ||
| + | :::Create 12 mM, 14 mM, 16 mM Imidazole binding buffers | ||
| + | :::Three seperate protein expressions were carried out (as done above) | ||
| + | :::Each pellet was suspended in one of the binding buffers and then the cells were lysed using the sonicator (as :::done before) | ||
| + | :::The supernatant of each different binding buffer was run through the Ni-column | ||
| + | |||
| + | :Jeremiah & Chris: | ||
| + | :::Transformation failed | ||
| + | :::Set up phusion for TET plasmid | ||
| + | :::Ligate the phusion products (TET and TBP+) | ||
==August 28== | ==August 28== | ||
| + | :Joe: | ||
| + | :::Redigestion of RFPEP-R by DpnI | ||
| + | :::PCR of SBP-R, RFP-R using longamp | ||
| + | |||
| + | :Michelle: | ||
| + | :::Purification of large scale expression of M2-BL21 glycerol stock + LB AMP-CM using a Nickel column. | ||
| + | :::Conducted SDS-PAGE Coomassie and Western Transfer Blot protocol, which continued until tomorrow. | ||
| + | |||
| + | :Justin and Dafne: | ||
| + | :::Western blot analysis of above protein purification | ||
| + | :::Western blot analysis was uninspiring, indicating the concentration change did not effect purification | ||
| + | |||
| + | :Jeremiah & Chris: | ||
| + | :::Run ligation on a gel (gel #176) | ||
| + | :::Gel showed no ligation occurred | ||
| + | :::Phusion PCR the TET plasmid and TBP+ gene (Long Amp Taq) | ||
| + | :::Ligate the phusion products | ||
| + | :::Transform into TOP 10 cells | ||
==August 29== | ==August 29== | ||
| + | :Michelle: | ||
| + | :::SDS-PAGE Coomassie staining of gel followed by desalting of protein at ~30kDa, which was successful. | ||
==August 30== | ==August 30== | ||
| + | :Joe: | ||
| + | :::PCR clean up - gel 173 good | ||
| + | :::Transform, plate on AMP | ||
| + | :Michelle: | ||
| + | :::Develop Western Transfer Blot of purified large scale expression of M2-BL21 glycerol stock + LB AMP-CM. | ||
| + | :::Modified ELISA assay using starch and testing with iodine to check the presence of starch in wells. Purified :::SBP-LRP also tested with Starch-ELISA. | ||
==August 31== | ==August 31== | ||
| + | :Joe: | ||
| + | :::Colony PCR of RFPEP-R | ||
Latest revision as of 01:42, 27 October 2012
Protocols | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Final Weeks |
Contents |
August 27
- Joe: fusion and transformation
- Michelle:
- Pellet large scale expression to prepare for purification of M2-BL21 glycerol stock
- + LB AMP-CM.
LJustin and Dafne:
- Determine optimal Imidazole concentration to utilize in Ni-column purification
- Create 12 mM, 14 mM, 16 mM Imidazole binding buffers
- Three seperate protein expressions were carried out (as done above)
- Each pellet was suspended in one of the binding buffers and then the cells were lysed using the sonicator (as :::done before)
- The supernatant of each different binding buffer was run through the Ni-column
- Jeremiah & Chris:
- Transformation failed
- Set up phusion for TET plasmid
- Ligate the phusion products (TET and TBP+)
August 28
- Joe:
- Redigestion of RFPEP-R by DpnI
- PCR of SBP-R, RFP-R using longamp
- Michelle:
- Purification of large scale expression of M2-BL21 glycerol stock + LB AMP-CM using a Nickel column.
- Conducted SDS-PAGE Coomassie and Western Transfer Blot protocol, which continued until tomorrow.
- Justin and Dafne:
- Western blot analysis of above protein purification
- Western blot analysis was uninspiring, indicating the concentration change did not effect purification
- Jeremiah & Chris:
- Run ligation on a gel (gel #176)
- Gel showed no ligation occurred
- Phusion PCR the TET plasmid and TBP+ gene (Long Amp Taq)
- Ligate the phusion products
- Transform into TOP 10 cells
August 29
- Michelle:
- SDS-PAGE Coomassie staining of gel followed by desalting of protein at ~30kDa, which was successful.
August 30
- Joe:
- PCR clean up - gel 173 good
- Transform, plate on AMP
- Michelle:
- Develop Western Transfer Blot of purified large scale expression of M2-BL21 glycerol stock + LB AMP-CM.
- Modified ELISA assay using starch and testing with iodine to check the presence of starch in wells. Purified :::SBP-LRP also tested with Starch-ELISA.
August 31
- Joe:
- Colony PCR of RFPEP-R
