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Team:Austin Texas/Week of July 9 to July 15
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== Design of Converter Plasmid == | == 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 == | == 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 == | == Spinach Aptamer == | ||
== CBB5 Caffeine Inducible Promoters == | == 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. | ||
| + | **7/11 - Miniprep of ORF1 - 1 and -2. Submit minipreped DNA for sequencing | ||
| + | **7/12 - Sequencing failed | ||
| + | **7/13 - Resubmitted sequencing with new primer stocks. | ||
== Inducible Odor == | == Inducible Odor == | ||
Latest revision as of 21:16, 31 July 2012
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.
- 7/11 - Miniprep of ORF1 - 1 and -2. Submit minipreped DNA for sequencing
- 7/12 - Sequencing failed
- 7/13 - Resubmitted sequencing with new primer stocks.
