IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/20: Difference between revisions

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***Biobrick ends were only found in:   
***Biobrick ends were only found in:   
***We will go back and possibly redo ligations/transformations  
***We will go back and possibly redo ligations/transformations  
*Diagnosed the difficulties we're having with mutagenesis:
<html><pre>
"c1" and "c2" are the customization (mutagenesis) sites corresponding to the capitalized portions of the primers suggested by the WMD3 tool. "c1" corresponds to the capitalized region of what WMD3 calls primer3 and to the reverse compliment of the capitalized region of primer4. Similarly, "c2" corresponds to the capitalized region of primer1 and the reverse complement of the capitalized region of primer2. For instance, WMD3 gives us the following when asked to design primers for the BET sequence:
primer1: gaTTGGTTATATATATCGCACCTtctctcttttgtattcc
primer2: gaAGGTGCGATATATATAACCAAtcaaagagaatcaatga
primer3: gaAGATGCGATATATTTAACCATtcacaggtcgtgatatg
primer4: gaATGGTTAAATATATCGCATCTtctacatatatattcct


In the sequence itself, the
File c1 Seen c2 Seen
----- First Sequencing Batch ------------
BETF-Fwd.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BETR-Rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
----- Second Sequencing Batch -----------
bet_2_f-forw TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
bet_3_f-forw TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
----- Third Sequencing Batch ------------
BET1F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET1R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET2F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET2R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET3F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET3R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET4F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET4R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET5F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET5R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET6F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
BET6R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
LTP1F-fwd.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
...
----- Theoretical Expectations ----------
RS300 Plasmid TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT
Bet.ape AGATGCGATATATTTAACCAT TTGGTTATATATATCGCACCT
GFP.ape GTACAGCTCGCCGTCCACTAT TTAGTGGTCGGCGAGCTGCAC
LTP.ape GTAGTCCTATACAAAGTTAGT TCTAACTATGTATAGGACCAC
RS300.ape TATGGACTGAAGGGGACCCGT ACAGGTCCCCTTCTGTCCATT
RS300 Backbone TATGGACTGAAGGGGACCCGT ACAGGTCCCCTTCTGTCCATT
</pre></html>
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Revision as of 12:47, 20 July 2010

iGEM iGarden <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Team Flavor

  • Picked colonies from Mira/Brazz StrepII STOP constructs
    • Only Mira N and Brazz C had colonies, 2 mL cultures were started and will be miniprepped to confirm ligation at the end of today.
  • Diluted Mira/Brazz YFP expression colonies 25:1 in LB+AMP for eventual IPTG induction. Will culture @ 37°C for 1 hour then take O/D's.

Team Allergy

  • Grew up colonies from transformations (all but BetS and PAL had colonies)
  • Checked on sequencing results of amiRNA parts sent in
    • The sequences did not match the ones that we wanted
      • The customization sequences that we wanted (our miRNA sequence+ a little bit of RS300) were not present in all of the LTP and Bet sequences sent in
      • Biobrick ends were only found in:
      • We will go back and possibly redo ligations/transformations
  • Diagnosed the difficulties we're having with mutagenesis:

<html><pre> "c1" and "c2" are the customization (mutagenesis) sites corresponding to the capitalized portions of the primers suggested by the WMD3 tool. "c1" corresponds to the capitalized region of what WMD3 calls primer3 and to the reverse compliment of the capitalized region of primer4. Similarly, "c2" corresponds to the capitalized region of primer1 and the reverse complement of the capitalized region of primer2. For instance, WMD3 gives us the following when asked to design primers for the BET sequence: primer1: gaTTGGTTATATATATCGCACCTtctctcttttgtattcc primer2: gaAGGTGCGATATATATAACCAAtcaaagagaatcaatga primer3: gaAGATGCGATATATTTAACCATtcacaggtcgtgatatg primer4: gaATGGTTAAATATATCGCATCTtctacatatatattcct

In the sequence itself, the

File c1 Seen c2 Seen ----- First Sequencing Batch ------------ BETF-Fwd.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BETR-Rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT ----- Second Sequencing Batch ----------- bet_2_f-forw TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT bet_3_f-forw TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT ----- Third Sequencing Batch ------------ BET1F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET1R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET2F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET2R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET3F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET3R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET4F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET4R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET5F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET5R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET6F-f.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT BET6R-rev.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT LTP1F-fwd.seq TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT ... ----- Theoretical Expectations ---------- RS300 Plasmid TTGGACTGAAGGGAGCTCCC GAGAGCTTCCTTGAGTCCAT Bet.ape AGATGCGATATATTTAACCAT TTGGTTATATATATCGCACCT GFP.ape GTACAGCTCGCCGTCCACTAT TTAGTGGTCGGCGAGCTGCAC LTP.ape GTAGTCCTATACAAAGTTAGT TCTAACTATGTATAGGACCAC RS300.ape TATGGACTGAAGGGGACCCGT ACAGGTCCCCTTCTGTCCATT RS300 Backbone TATGGACTGAAGGGGACCCGT ACAGGTCCCCTTCTGTCCATT </pre></html>


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