GROUP 4: Long Two-way Stops & Mat(alpha) specific promoters
Summary: As of September 23rd, 2008
We had successfully cloned BBa_K110005 and BBa_K110006 (alpha Promoters) and
BBa_K110003 (Long Terminator) into pGEM-T and Kanamycin biobrick vectors.
They await assembly and ligation.
Date: September 17th, 2008
Minipreps were made of the kanamycin biobrick vector clones, and digested with E/P to
check for insert of correct size. See gel: /pic//
Low-copy origin makes for poor gel visualization. Suggest some other diagnostic test.
Date: September 12th, 2008
Clones were digested with E/P, and ligated O/N into Kanamycin biobrick vector (pSB4K5).
Colonies were picked and grown.
Date: August 26th, 2008
Author: Jonathan
Sequences were returned, and prefix/suffix/identities were confirmed. Clones that passed are:
Long Terminator BBa_K110003: Clone A1-3.2
Alpha Promoter BBa_K110005: Clone 5.1
Alpha Promoter BBa_K110006: Clones 6.1, 6.3
Date: August 21st, 2008
Author: Jonathan
Clones were grown up O/N and re-miniprepped. Digestion with E/P yielded some possible candidates.
Here is the gel pic: [http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-10-29.08/21/08%20E/P%20Restriction%20Digest%20for%20BBs%203,%205,%206%20(pGEM-T).Alpha%20Promoters/Long%20Terminators%20Restriction%20Digest.html 08/21/08 E/P Restriction Digest for BBs 3, 5, 6 (pGEM-T)]
Clones submitted for sequencing.
Date: August 1st, 2008
Author: Jonathan
Sequences were submitted to the facility (3/building block) and a few returned perfect clones, but
due to miscommunication, exactly which clones that the trace files correspond to are under some
confusion. Will attempt to confirm clone identities.
Date: July 29, 2008
Status report by: Jaime
Part no.: BBa_K110001, BBa_K110003, BBa_K110005, BBa_K110006
Part Description: Long Two-way Stops & Mat(alpha) specific promotors
[http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-7-25.Restriction%20Enzyme%20Digest%20of%20Mini-Preps.Jaime.html Restriction Enzyme Digest of Mini-Preps]
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
[http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-7-22.iGEM%20PCR%20experimentation%20on%20BBa_K110001%20and%20BBa_K110003.Jaime.html iGEM PCR experimentation on BBa_K110001 and BBa_K110003]
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
[http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-7-22.iGEM%20PCR%20experimentation%202%20on%20BBa_K110001%20and%20BBa_K110003.Jaime.html iGEM PCR experimentation 2 on BBa_K110001 and BBa_K110003]
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
[http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-7-22.iGEM%20BBa_K110005%20and%20BBa_K110006.Jaime.html iGEM BBa_K110005 and BBa_K110006]
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
[http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-7-22.iGEM%20BBa_K110005%20and%20BBa_K110006.Jaime.html iGEM BBa_K110005 and BBa_K110006]
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
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