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 = Good News
 * .1 &mu;L IAA and .5 &mu;L IAA smelled strongly and pleasantly of bananas in IK strains. It is reasonable to estimate that our ATF1 gene can generate in E. coli about this range of IAA
 * IK gave a huge improvement over the Top10 smell cultures

Indole Knockouts
Out of 3 strains, the JC12337 did not smell as nice Made glycerols of MB408 and MEB61
 * note, previous disappointment with IK stemmed from contaminated LB

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

 * 1) Cut ATF1A* with EcoRI and XbaI 38 ng/&mu;L
 * 2) Cut R0030 with EcoRI and SpeI 10 ng/&mu;L
 * 3) Cut R0032 with EcoRI and SpeI 23 ng/&mu;L

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