Team:Johns Hopkins/Notebook

From 2008.igem.org

(Difference between revisions)
(Important reminders and notes)
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   therefore not optimal. Advice/help on this issue would be appreciated.
   therefore not optimal. Advice/help on this issue would be appreciated.
-
=== GROUP 2: MATa Specific-promoters ===
+
=== [[Team:Johns Hopkins/Notebook/GROUP 2: MATa Specific-promoters]] ===
   Status report by Allison and Nate
   Status report by Allison and Nate
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   (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1478)</b>
   (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1478)</b>
-
=== GROUP 3: Short two way stops ===
+
=== [[Team:Johns Hopkins/Notebook/GROUP 3: Short two way stops]] ===
   Date: 7/21/08
   Date: 7/21/08
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   Current status of this part:
   Current status of this part:
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=== GROUP 4: Long Two-way Stops & Mat(alpha) specific promotors ===
+
=== [[Team:Johns Hopkins/Notebook/GROUP 4: Long Two-way Stops & Mat(alpha) specific promotors]] ===
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   sequencing- put in incubator on 7/15
   sequencing- put in incubator on 7/15
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=== GROUP 5: MATa Specific Promoters II ===
+
=== [[Team:Johns Hopkins/Notebook/GROUP 5: MATa Specific Promoters II]] ===
   Date: 7/11/08
   Date: 7/11/08
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   Current status of this part: This part must be restriction enzyme digested and sequenced <br> next
   Current status of this part: This part must be restriction enzyme digested and sequenced <br> next
-
=== GROUP 6: Vectors ===
+
=== [[Team:Johns Hopkins/Notebook/GROUP 6: Vectors]] ===
   Status report by ____
   Status report by ____
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   Preparative digests ready for use are located – where?
   Preparative digests ready for use are located – where?
-
=== GROUP 7: Microscopy/Yeast ===
+
=== [[Team:Johns Hopkins/Notebook/GROUP 7: Microscopy/Yeast]] ===
   Milestones  
   Milestones  

Revision as of 00:56, 23 July 2008

Contents

Groups

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]
 
 July 11: Primers for group 1 were delivered yesterday.
 July 11: Lab meeting at 7:30PM in the lab to go over miniprep protocol.
 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

Summary for Fluorescent Proteins Group

 Date: July 22, 2008
 Status report by: Tejas
 Part no.: BBa_K110017 -> BBa_K110023
 Part Description: yESapphire , mCherry, venusYFP, and Citrine
 
  Work on yESapphire was gratiously done by James. Primers were designed. Restriction site ends
 have been added through PCR. The product was cloned into JM109. Colonies were picked and an
 inoculation was grown. Miniprep was performed and subsequent DNA was CS PCR'd and run on a gel
 to verify contents. BioBrick is currently being sequenced.
 
  Work on mCherry and venusYFP (BBa_K110018 -> BBa_K110021) is currently being done. Primers were
 designed. Restriction sites have been added through PCR. The products were cloned into JM109 and
 plated. 3 colonies per 4 BioBricks (total 12) were picked, grown out, miniprepped, and digested
 for verification on a gel.
  According to the results, ([http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.Venus%20YFP%20and%20mCherry%20Miniprep%20check%20via%20digest.Ingrid.html Venus YFP and mCherry Miniprep check via digest]), only one of the
 mCherry's (BBa_K110019) is the correct product. This product will be retransformed to yield more
 for sequencing. Colonies for the other three BioBricks were selected by Ingrid on 7.22.2008 and
 are being grown out. Process will continue as per protocol.
 
  Work on Citrine has not yet begun. Primers have been designed and ordered, but template DNA must
 be grown out. Template DNA to be grown and extracted by James should we later decide that Citrine
 will be needed.
 
 *Note that mCherry and Citrine both have restriction sites within the coding region, and are
 therefore not optimal. Advice/help on this issue would be appreciated.

Team:Johns Hopkins/Notebook/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)</b>
 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)

Team:Johns Hopkins/Notebook/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:

Team:Johns Hopkins/Notebook/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

Team:Johns Hopkins/Notebook/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

Team:Johns Hopkins/Notebook/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?

Team:Johns Hopkins/Notebook/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