Jessica Karen Wong/Notebook/2007-7-2
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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