IGEM:MIT/2006/Notebook/2006-7-25

IAA Smell Test

Added .1, .5, 1, 5, 10ul to 5ml liquid cultures of Top10 .5ul was detectable, but .1ul (which we might get) was less detectable

• Tested today in preferred IK strains

Indole Knockouts

Out of 3 strains, the JC12337 did not smell as nice

• note, previous disappointment with IK stemmed from contaminated LB

Made glycerols of MB408 and MEB61

osmY-Trunc Eco/Spe Digest

• 5 uL Buffer 2
• 0.5 uL BSA
• 2 uL ~800 ng/uL Annealed osmY-Trunc primers
• 1.25 uL EcoRI
• 1.25 uL SpeI

ATF1A, R0030, R0032 Digests

Cut ATF1A with EcoRI and XbaI ---- 38 ng/μL Cut R0030 with EcoRI and SpeI ---- 10 ng/μL Cut R0032 with EcoRI and SpeI ---- 23 ng/μL

Order ATF1-A mutation primers

Primer pair 2

                                   *
   Forward: 5' CACCCCCTGGATAAGCGAATTTGACATGAATGATAACAAAG 3'
   Reverse: 5' CTTTGTTATCATTCATGTCAAATTCGCTTATCCAGGGGGTG 3'
                                   *
    GC content: 41.46%           Location: 214-254
    Melting temp: 79.8°C         Mismatched bases: 1
    Length: 41 bp                Mutation: Substitution
    5' flanking region: 20 bp    Forward primer MW: 12619.37 Da
    3' flanking region: 20 bp    Reverse primer MW: 12587.31 Da