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Week 13 (11/29 – 12/23)
Inhibition Assays
TUESDAY: Materials & Methods for Enzyme & Inhibition Assays – upload this to the Google Docs as a PDF. See ‘Final Report‘ category on blog
Final Report Due Sat. Dec 4th
Lab Notebook Due Sat. Dec 4th
Class Party @ Kathryn’s on Sunday Dec. 5th (7pm)
Future
MILESTONES:
gene cloned into pNIC-Bsa4, protein expressed in BL21(DE3), protein purified, characterized on gel and concentrated, complete virtual screen jobs on libraries in DatabasesVDS, order compounds, test if enzyme is functional (Enzyme Assay), test compounds in Inhibition assay, determine IC50 of good compounds
Final Report Due Sat. Dec 4th
Week 13 (11/22 – 11/28)
Inhibition Assays
1st Week
continue cloning into pNIC-Bsa4
Weekly update to WordPress blog
2nd Week
Finish pNIC-Bsa4 cloning by end of week
Tues & Wed: PyMol Refresher – upload to GDocs
Tues & Wed: Virtual Screen Refresher
Print out of TxClass Safety Training – N/A already done for Vets
Weekly update to WordPress blog due on Sunday
3rd Week (9/13 – 9/19)
PyMol Refresher – post to GDocs
Virtual Screen Refresher – post to GDocs and post table of top 10 results to Blog
Continue pNIC-Bsa4 cloning
Verify DNA sequence of successful clone
Maxi prep successful clone
Friday: Lab Notebook check
Weekly update to WordPress blog due on Sunday
Week 4 (9/20 – 9/26)
Tues: First journal club
ProteinExpressionSCALE_UP_VDS
ProteinPurificationVDS
ProteinCharacterizationVDS
Weekly update to WordPress blog due on Sunday
Sunday: ROUGH DRAFT of Research Report on Target Enzyme
Week 5 (9/27 – 10/3)
Finish cloning of your target into pNIC-Bsa4
Start Virtual Screening of Your Target
Weekly update to WordPress blog due on Sunday – should include image of results
Week 6 (10/4 – 10/10)
Finish cloning of your target into pNIC-Bsa4
verify positive clones with RE digest and DNA Sequencing (must virtually reconstruct your DNA first)
Transform into BL21(DE3) cells
Start expression of target
Continue Virtual Screening of Your Target
Sunday 10/10 : FINAL DRAFT of Research Report on Target Enzyme
Weekly update to WordPress blog due on Sunday – should include an image of results
Week 7 (10/11 – 10/17)
verify positive clones with RE digest and DNA Sequencing (must virtually reconstruct your DNA first)
Transform into BL21(DE3) cells
Start expression of target
Examine first Virtual Screening Run of Your Target
Continue screening the other libraries in the DatabasesVDS directory
Friday 10/15 – Lab Notebook Check
Weekly update to WordPress blog due on Sunday – should include an image of results
PyMol Image Contest – upload pic – Due: Tues Oct. 19th
Week 8 (10/18 – 10/24)
PyMol Image Contest – upload pic – Due: Tues Oct. 19th
verify positive clones with RE digest and DNA Sequencing (must virtually reconstruct your DNA first – tricky to do. Seek help from Dr. B or mentor) – must show how you did this in your lab notebook
Transform into BL21(DE3) cells
Start expression of target (strategize with Dr. B or mentor first)
Continue screening the other libraries in the DatabasesVDS directory – complete all by Halloween
Begin enzyme assay tests
Weekly update to WordPress blog due on Sunday – should include an image of results
Week 9 (10/25-10/31)
If you have not obtained a positive clone of your gene, then we need to go ahead and fast-track you to protein expression. To do this, you need to obtain an expression plasmid with your gene from Dr. B.
Ideally Wednesday and Friday could be used to do the actual expression. Since one of the steps is in the middle of the day, we will have the mentors do that part for you if you have class then. However, you will need to setup the transformation and overnight expression steps on your own beforehand (Monday and Tuesday – Ideally) and do the Spin down in the evening. A tentative schedule is listed below.
See the GDocs for the full protocol: ProtocolProteinExpressionScaleUpVDS_fall10.doc
Kimberly: keep working on your own protein expression schedule or jump in with this if you need to
Gabriela: continue working on cloning since you have done some protein expression and assays already
What: TRANSFORMATION into BL21(DE3)
When: MONDAY (preferably)- 1.5 hrs:
Who: should do this:
- Damir (since your plasmid did not seem to express well)
- Radhika
- Yiling (if yours doesn’t grow up at all)
- Da’Marcus
What: OVERNIGHT GROWTH in 50 ml of LB
When: Tuesday evening (20 minutes to set up if you have LB), also prepare large Erlenmeyer flasks with LB for the next day when the mentor will grow up your samples.)
Who: should do this:
- Damir
- Radhika
- Yiling
- Da’Marcus
What: Coordinate with Adam and Kathryn to read OD600 of your Overnight growth and transfer to large flasks during the day. Then spin down your expressed cells (with Dr. B in the evening)
When: Wednesday (midday – mentors express, ~ 6:00 -8:00 students spin down)
Who: should do this:
- Damir
- Radhika
- Yiling
- Da’Marcus
After expression – proceed to sonication, purification and characterization.
ALternatively, these can be all shifted by a day or so in order to do Protein Expression on Friday.
PyMol Image Contest voting
FRI External Open House – Oct 28th 1-4 pm
Continue screening the other libraries in the DatabasesVDS directory – complete all by Halloween
Weekly update to WordPress blog due on Sunday – should include an image of results
Week 10 (11/1-11/7)
FINISH virtual Screening – Do the Top Ligands protocol – This is due by Tuedsday NIGHT.
Continue with protein expression, purify, characterize on protein gel, concentrate protein, dialysis (?)
If you need to express again – coordinate with Dr. B and Mentors
Kimberly
Damir
Radhika
Yiling
Da’Marcus
Thao
Gabriela: continue working on cloning since you have done some protein expression and assays already
Weekly update to WordPress blog due on Sunday – should include an image of results
TUESDAY: Materials & Methods for Cloning is due (this will be used for your Final Report) – upload this to the Google Docs as a PDF.
see post: https://vdsclass.wordpress.com/2010/11/03/mm-for-cloning/
Week 11 (11/8-11/14)
TUESDAY: Materials & Methods for Cloning is due (this will be used for your Final Report) – upload this to the Google Docs as a PDF.
Characterize on protein gel, concentrate protein
Start Enzyme Assays (variables: enzyme amount, substrate amount, DMSO tolerance)
Weekly update to WordPress blog due on Sunday – should include an image of results
NEXT TUESDAY: Materials & Methods for Virtual Screening is due – upload this to the Google Docs as a PDF.
see post: https://vdsclass.wordpress.com/2010/11/08/mm-for-cloning/
Week 12 (11/15-11/21)
Enzyme Assays (variables: enzyme amount, substrate amount, DMSO tolerance)
Inhibition Assays
TUESDAY: Materials & Methods for Virtual Screening is due – upload this to the Google Docs as a PDF. See ‘Final Report‘ category on blog
Weekly update to WordPress blog due on Sunday – should include an image of results
NEXT TUESDAY: Materials & Methods for Expression, Purification, Characterization is due – upload this to the Google Docs as a PDF. See ‘Final Report‘ category on blog
Week 13 (11/22 – 11/28)
Continue Enzyme Assays (variables: enzyme amount, substrate amount, DMSO tolerance)
Inhibition Assays
TUESDAY: Materials & Methods for Expression, Purification, Characterization is due – upload this to the Google Docs as a PDF. See ‘Final Report‘ category on blog
Weekly update to WordPress blog due on Sunday – should include an image of results
NEXT TUESDAY: Materials & Methods for Enzyme & Inhibition Assays – upload this to the Google Docs as a PDF. See ‘Final Report‘ category on blog
VDSclass Research Collaborative 
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