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Team:Austin Texas/Week of July 9 to July 15
From 2012.igem.org
Contents |
Notebook for week of July 9 to July 15
Design of Converter Plasmid
- Razan
- 7/9- Gibson Assembly of Converter and Control plasmids
- 7/10- Transformation of gibson failed and gel shows no correct bands
- 7/11- Redid Gibson assembly of Converter and Control plasmids with longer incubation time; same results.
Design of Reporter Plasmid
- Ben
- Made 5 uM stocks of all pReporter primers
- PCR:
- pIGM023 (pET21.KOD-BB): JE.pET.BB.F, JE.pET.BB.R
- pIGM022 (pAK.TAQ-rrnB): JE.rrnB.F, JE.rrnB.R
- pIGM006 (pSB1A2-K081009 [LasI]): JE.LasI.F, JE.LasI.R
- pIGM005 (pSB1AK3-K081014 [RFP]): BS.RFP.F, BS.RFP.R
- pIGM025 (pACYC.T7.GFP-sfGFP): BS.sfGFP.F, JE.sfGFP.R *Note*: used incorrect template. Will use pIGM026.
- pIGM008 (pSB1AK3-B0015 [double.terminator]): JE.GFPTerm.F, JE.GFPTerm.R
- Results: No prepped plasmids available as template for IGM005-6, so I tried using BioBrick transforming DNA as template; these rxns didn't work. Will try again with prepped DNA.
- Made test tube cultures with IGM004 (pSB3K3-I714891 [GFP]), IGM005 (pSB1AK3-K081014 [RFP]), and IGM006 (pSB1A2-K081009 [LasI]) for the retry.
- Prepped DNA.
- PCR: (retry of previous with new templates)
- pIGM023 (pET21.KOD-BB): JE.pET.BB.F, JE.pET.BB.R
- pIGM022 (pAK.TAQ-rrnB): JE.rrnB.F, JE.rrnB.R
- pIGM006 (pSB1A2-K081009 [LasI]): JE.LasI.F, JE.LasI.R
- pIGM005 (pSB1AK3-K081014 [RFP]): BS.RFP.F, BS.RFP.R
- pIGM026 (pET21-sfGFP): BS.sfGFP.F, JE.sfGFP.R
- pIGM008 (pSB1AK3-B0015 [double.terminator]): JE.GFPTerm.F, JE.GFPTerm.R
- Results: All lanes contained correct-size bands.
- Cleaned up DNA.
- OE-PCR:
- BS.LoxLac.F1 + BS.LoxLac.F2 + BS.LoxLac.R1
- BS.LoxLacFlipped.F1 + BS.LoxLacFlipped.F2 + BS.LoxLacFlipped.R1
- Results: Accidentally used 10X excess insert instead of 10X excess of outer primers. Will retry with correct conditions.
- OE-PCR:
- BS.LoxLac.F1 + BS.LoxLac.F2 + BS.LoxLac.R1
- BS.LoxLacFlipped.F1 + BS.LoxLacFlipped.F2 + BS.LoxLacFlipped.R1
- Results: All lanes contained correct-size bands.
- Gel-extracted LoxLac and LoxLacFlipped sequences.
- PCR:
- LoxLac: BS.LoxLac.F1, BS.LoxLac.R1
- LoxLacFlipped: BS.LoxLacFlipped.F1, BS.LoxLacFlipped.R1
- Results: All lanes contained correct-size bands.
- Cleaned up DNA.
- OE-PCR:
- rrnB + LasI: JE.rrnB.F, JE.LasI.R
- RFP + LoxLac: BS.RFP.F, BS.LoxLac.R1
- RFP + LoxLacFlipped: BS.RFP.F, BS.LoxLacFlipped.R1
- sfGFP + double.terminator: BS.sfGFP.F, JE.sfGFP.R
- Results: All lanes contained correct-size bands except for RFP + LoxLacFlipped. Will retry this rxn.
- OE-PCR:
- RFP + LoxLacFlipped: BS.RFP.F, BS.LoxLacFlipped.R1
- Results: All lanes contained correct-size bands.
- PCR:
- rrnB.LasI
- RFP.LoxLac
- RFP.LoxLacFlipped
- sfGFP.double.terminator
- Results: All lanes contained correct-size bands.
- Cleaned up DNA, ran on gel, and gel extracted.
- Dpn1 digested pET21.BB amplicon. Cleaned up.
- Gibson assembly:
- pET21.BB (control)
- pET21.BB + rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator
- pET21.BB + rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator
- Results: No visible product band.
- Desalted, transformed, and plated on LB+Cab.
Spinach Aptamer
CBB5 Caffeine Inducible Promoters
- Peter
- 7/9 - Set up confirmation PCR of cultures. 1 for each culture, as well as 2 controls from CBB5 genomic DNA.
- 7/10 - Gel electrophoresis of confirmation PCR showed 2 successful Gibson product colonies, as well as both successful controls from CBB5 genomic DNA.
