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Team:Johns Hopkins/Notebook
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iGEM Groups 1.0
iGEM Groups 2.01
iGEM Groups 2.02
Important reminders and notes
[Can make general comments here, so they don't get lost in peoples e-mail boxes]
[For anyone not part of the iGEM team who would like to see the gels we're running: 1. Click the links provided 2. Guest login: bluejays
July 11: Primers for group 1 were delivered yesterday
July 11: Lab meeting at 7:30PM in the lab to go over miniprep protocol
Tuesday July 15: Lab meeting at 6:30PM with Jessica. Have status reports ready. Bring labtop if you can
July 17: Restriction Digest/Sequencing Preparation (with James) 6:00PM
July 21: 6:00PM or 6:30PM Lab meeting with Jessica. Have status reports ready.
Status Reports
The status reports of each group below will continuously be updated as we work on the biobricks. The following PDFs contain progressive versions of our status reports as we continue through the sex detector project; they are added weekly. To learn more about each biobrick, please refer to the Biobrick page.
Status Report 2.1 - 07/12/08
Status Report 2.2 - 07/17/08
Please BOLD the most recent step that you have completed. Do this by placing the tag < b > in front of and </ b > at the end of what you would like to be placed in bold (with no space between letter and carrot symbol (<,>).
GROUP 1: Fluorescent Proteins
Date: July 1 2008 status report by: Ingrid (work done by James) Part no.: BBa_K110017 Part Description: yESapphire Part Location (in build a genome lab): In James and Jasper's PCR product Box, Stainless Steel 4 degree PCR successful?; Yes Cloning of PCR product successful: Y/N Sequencing of cloned PCR product successful: Not done Joining of validated part to adjacent part(s) status: Not done Current status of this part: This part is being Sequenced
Date: July 1 2008 status report by: Ingrid (work done by James) Part no.: BBa_K110010 Part Description: yESapphire Part Location (in build a genome lab): In James and Jasper's PCR product Box, Stainless Steel 4 degree PCR successful?; Yes Cloning of PCR product successful: Y/N Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Current status of this part: This part is being sequenced
Date: July 17 2008 status report by: Ingrid Part no.: BBa_K110018 Part Description: mCherry: Part Location (in build a genome lab): In silver fridge by door PCR successful?; Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471) Cloning of PCR product successful: Inconclusive http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476 Sequencing of cloned PCR product successful: Not done Joining of validated part to adjacent part(s) status: Not done Problems to be solved: Had some extra unknown products, with unknown bands in the gel Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
Date: July 17 2008 status report by: Ingrid Part no.: BBa_K110019 Part Description: mCherry: Part Location (in build a genome lab): In silver fridge by door PCR successful?; Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471) Cloning of PCR product successful: Inconclusive http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476 Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Problems to be solved: Need to figure out if gel after digest shows correct products
Date: July 17 2008 status report by: Ingrid Part no.: BBa_K110020 Part Description: Venus Enhanced YFP Part Location (in build a genome lab): In silver fridge by door PCR successful?; Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471) Cloning of PCR product successful: inconclusive http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476 Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Problems to be solved: Had some extra unknown products, with unknown bands in the gel Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
Date: July 17 2008 status report by: Ingrid Part no.: BBa_K110021 Part Description: Venus Enhanced YFP Part Location (in build a genome lab): In silver fridge by door PCR successful?; Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471) Cloning of PCR product successful: Inconclusive http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476 Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Problems to be solved: Had some extra unknown products, with unknown bands in the gel Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
GROUP 2: MATa Specific-promoters
Status report by Allison and Nate Part no.: BBa_K110008 Part Description: MFA1 (L+R) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to front door Date: 7/10/08 PCR successful? Yes http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&advanced=0&paging=&page=33 Cloning of PCR product successful: in progress Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: to be determined Current status of this part: PCR was being troubleshooted, appeared to have good results with regular PCR protocol (not touchdown) in which there was a constant annealing temperature of 55 degrees C - see gel
Status report by Allison and Nate Part no.: BBa_K110016 Part Description: Ste2 (R+L) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to front door Date: 7/10/08 PCR successful? Yes http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&advanced=0&paging=&page=33 Cloning of PCR product successful: in progress Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: to be determined Current status of this part: Both PCR protocols (touchdown and second PCR with constant annealing temperature) produced product of the correct size. BBa_K110016 was used as a control in the second PCR with BBa_K110008.
Status report by Allison and Nate Part no.: BBa_K110008 Part Description: MFA1 (L+R) Part Location: same as above, plates are at 4 degrees refrigerator near front door Date: 7/14/08 PCR successful? Yes Cloning of PCR product of successful? There were mainly light blue colonies (only a couple white colonies) Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: Ligation Current status of this part: plates are at 4 degrees; another ligation/transformation will be completed soon
Status report by Allison and Nate Part no.: BBa_K110016 Part Description: Ste2 (R+L) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to front door; plates at 4 degrees Date: 7/14/08 PCR successful? Yes Cloning of PCR product successful: There were many blue colonies (similar to the plate of BB_K110008) Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: Ligation Current status of this part: plates are at 4 degrees; another ligation/transformation will be completed soon
Status report by Allison and Nate Part no.: BBa_K110008 Part Description: MFA1 (L+R) Part Location: same as above, plates are at 4 degrees refrigerator near front door Date: 7/22/08 PCR successful? Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&mode=single&page=2 Cloning of PCR product of successful? Yes (approx 60 white colonies between two plates) Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: Current status of this part: 12 mini preps and the restriction digestion were completed; preparing to send 3 samples to be sequenced (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1478)
Status report by Allison and Nate Part no.: BBa_K110016 Part Description: Ste2 (R+L) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to front door; plates at 4 degrees Date: 7/22/08 PCR successful? Yes Cloning of PCR product successful: Yes (approx 20 white colonies on one plate) Joining of validated part to adjacent part(s) status: not done Problems to be solved: Current status of this part: 12 mini preps and the restriction digestion were completed; preparing to send 3 samples to be sequenced (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1478)
GROUP 3: Short two way stops
Date: 7/21/08 status report by: James Part no.: BBa_K110011 Part Description: Between-bud 27-W FRS2-C LtR Part Location (in build a genome lab): In James and Jasper's PCR product Box, Stainless Steel 4 degree PCR successful?; Yes Cloning of PCR product successful: Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1475) Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Problems to be solved: Current status of this part: Miniprep digestion yielded no product, PCR; ligation; transformation; and miniprep will be repeated by this Wednesday (07/23/08)
status report by: James Part no.: BBa_K110012 Part Description: Between STE2-W and BST1-C LtR Part Location (in build a genome lab): In James and Jasper's PCR product Box, Stainless Steel 4 degree PCR successful?; Yes Cloning of PCR product successful: Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1475) Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Problems to be solved: Current status of this part: Miniprep digestion yielded no product, PCR; ligation; transformation; and miniprep will be repeated by this Wednesday (07/23/08)
status report by: James Part no.: BBa_K110013 Part Description: Between-SWP82-W and EMP47-C LtR Part Location (in build a genome lab): In James and Jasper's PCR product Box, Stainless Steel 4 degree PCR successful?; No Cloning of PCR product successful: No Sequencing of cloned PCR product successful: No Joining of validated part to adjacent part(s) status: Not done Problems to be solved: The PCR of this part yielded a very large product Current status of this part:
GROUP 4: Long Two-way Stops & Mat(alpha) specific promotors
Date: 7/10/08
Status report by: Jaime Liu Part no.: BBa_K110001 Part Description: BBa_K110001 - Between-bud 27-W FRS2-C + 200bp into each gene LtR Part Location (in build a genome lab): In 4C fridge #2 PCR successful?; Y/N (link such as this)- Yes BBa_K110001: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1462 Cloning of PCR product successful: Y/N Yes Sequencing of cloned PCR product successful: Y/N No Joining of validated part to adjacent part(s) status: Not Done Problems to be solved: Not really a problem, but need do a mini-Prep and sequence Current status of this part: All cloned and inoculated into 100uL in 96 well plate for sequencing- put in incubator on 7/15
Date: 7/10/08
Status report by: Jaime Liu Part no.: BBa_K110003 Part Description: BBa_K110003 - Between-SWP82-W and EMP47-C +200 into each gene LtR Part Location (in build a genome lab): In 4C fridge #2 PCR successful?; Y/N (link such as this)- Yes BBa_K110003: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1463 Cloning of PCR product successful: Y/N Yes Sequencing of cloned PCR product successful: Y/N No Joining of validated part to adjacent part(s) status: Not Done Problems to be solved: Not really a problem, but need do a mini-Prep and sequence Current status of this part: All cloned and inoculated into 100uL in 96 well plate for sequencing- put in incubator on 7/15
Date: 7/10/08
Status report by: Jaime Liu Part no.: BBa_K110005 Part Description: BBa_K110005 - MFalpha2 LtR Part Location (in build a genome lab): In 4C fridge #2 PCR successful?; Y/N (link such as this)- Yes BBa_K110005: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1470 Cloning of PCR product successful: Y/N Yes Sequencing of cloned PCR product successful: Y/N No Joining of validated part to adjacent part(s) status: Not Done Problems to be solved: Not really a problem, but need do a mini-Prep and sequence Current status of this part: All cloned and inoculated into 100uL in 96 well plate for sequencing- put in incubator on 7/15
Date: 7/10/08
Status report by: Jaime Liu Part no.: BBa_K110006 Part Description: BBa_K110006 - MFalpha1 LtR Part Location (in build a genome lab): In 4C fridge #2 PCR successful?; Y/N (link such as this)- Yes BBa_K110006: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1470 Cloning of PCR product successful: Y/N Yes Sequencing of cloned PCR product successful: Y/N No Joining of validated part to adjacent part(s) status: Not Done Problems to be solved: Not really a problem, but need do a mini-Prep and sequence Current status of this part: All cloned and inoculated into 100uL in 96 well plate for sequencing- put in incubator on 7/15
GROUP 5: MATa Specific Promoters II
Date: 7/11/08 status report by Rick Carrick Part no.: BBa_K110015, Part Description: MFA1 Part Location (in build a genome lab): PCR successful?; Y (on moodle somewhere) Cloning of PCR product successful: Y Sequencing of cloned PCR product successful:N Joining of validated part to adjacent part(s) status: Not done Problems to be solved: None so far Current status of this part: This parts must be restriction enzyme digested and sequenced next.
Date: 7/11/08 status report by Rick Carrick Part no.: BBa_K110009 Part Description: Ste2 Part Location (in build a genome lab): PCR successful?; Y (on moodle somewhere) Cloning of PCR product successful: Y Sequencing of cloned PCR product successful:N Joining of validated part to adjacent part(s) status: Not done Problems to be solved: None so far Current status of this part: This part must be restriction enzyme digested and sequenced
next
GROUP 6: Vectors
Status report by ____ Vector transformed into bacteria strain DB3.1 Y/N Permanent culture made in the Boeke lab for future reference Y/N Selectable marker for this vector Medium made and tested Y/N (link) DNA preps made Y/N DNA preps tested by RE digest - (link) DNA preps tested by transformation into DB3.1 and DH5alpha (or JM109) – put data as Table on moodle. Sample format/data follows:
Amount transformed cfu/micGm in DB3.1 cfu/micGm in JM109 0.1 ng 5 * 10e7 <2 * 10e2
Preparative digests ready for use are located – where?
GROUP 7: Microscopy/Yeast
Milestones Have gfp yeast been visualized by microscopy? Have gfp yeast been photographed? Link to moodle – picture. [If pics are nice, put on our web page] Can green(/other colors) colonies be photographed? Has STE3-gfp sex detector been transformed into MATa, MATalpha, and MATa/alpha? Y/N Have permanent cultures been banked in the Boeke lab? Y/N Have STE3-gfp sex detector cells/colonies been photographed Y/N - link to Moodle
