Jessica Karen Wong/Notebook/2007-7-2

To Do

 * Retransform and plate RBS tester w/ RFP
 * Sequence promoter tester w/ RFP
 * Get part numbers for both
 * design BB primers
 * PCR T9002 and I2055

Registry

 * The RBS tester with RFP (that we used to call blue) is I2056
 * The promoter tester with RFP (used to call green) is I2057
 * Nishant sent I2057 for sequencing
 * To Recap: I2055 is the GFP RBS tester, E0240 is the GFP promoter tester, and T9002 is the output measurement tester (aka inverter, terminator, etc)

BioBrick Primers

 * E0240 needs a BioBrick site b/t the plasmid and the RBS
 * Forward Primer should match B0032 and have a Spe1 tail
 * Reverse Primers should have an EcoR1 tail and 1 should match 3K3 and the other 1AK3
 * I2055 needs a BioBrick site between the promoter and GFP
 * Fwd should match GFP (E0040) and have a Spe1 tail
 * Rev should match R0040 and have an EcoR1 tail
 * T9002 needs a BioBrick site between F2620 and B0032
 * Fwd should match B0032 and have Pst1 tail
 * Rev match Lux pR of F2620 and have EcoR1 tail

To insert BB sites. Format: 8-mer.restriction site.primer

* E0240: o F (incl E0240, PstI): CTTAGTAG.ACTAGT.TCACACAGGAAAGTACTAGATGCG (53.1) o R (incl 3K3/1AK3, EcoRI): TCAGCGAT.GAATTC.CAGAAATCATCCTTAGCGAAAGC (54.2)

* I2057: o F (incl I2057, PstI): CTTAGTAG.ACTAGT.TCACACAGGAAAGTACTAGATGGC (52.1) o R: same as E0240

* I2055: o F (incl GFP, SpeI): CTTAGTAG.ACTAGT.ATGCGTAAAGGAGAAGAACTTTTC (52.4) o R (incl R0040, EcoRI): TCAGCGAT.GAATTC.GTGCTCAGTATCTCTATCACTGATAGG (52.0)

* I2056: o F (incl RFP, SpeI): CTTAGTAG.ACTAGT.ATGGCTTCCTCCGAAGACG (54.0) o R: Same as I2055

* For T9002: o F (incl lux pR, PstI): TCAGCGAT.CTGCAG.TCACACAGGAAAGTACTAGATGCG (53.1) o R (incl RBS, GFP,EcoRI) : CTTAGTAG.GAATTC.TTTATTCGACTATAACAAACCATTTTC (51.9)

PCR

 * We did a 100ul preparatory PCR on T9002 at 54.5C
 * PCRed I2055 on a gradient 41-51
 * Diluted the new primers to 40uM
 * I2055 shorter F - 918 ul water
 * I2055 shorter R - 801 ul
 * I2055 longer R - 867.5 ul
 * T9002 R GFP - 801 ul