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Team:Austin Texas/Week of July 16 to July 22
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== Design of Reporter Plasmid == | == Design of Reporter Plasmid == | ||
| + | |||
| + | * Ben | ||
| + | ** '''Colony PCR:''' | ||
| + | *** pReporter: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** '''Results:''' No correctly sized bands. Will retry Gibson assembly with new master mix. | ||
| + | ** '''Gibson assembly:''' | ||
| + | *** pET21.BB (control) | ||
| + | *** pET21.BB + rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator | ||
| + | *** pET21.BB + rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator | ||
| + | *** '''Results:''' Still no visible product band. Will retry Gibson assembly after OE-PCRing each set of inserts into 1 piece first. | ||
| + | ** '''OE-PCR:''' | ||
| + | *** rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R '''*Note*: These last two rxns did not have primers added until after 15 cycles.''' | ||
| + | *** '''Results:''' All lanes contained correct-size bands. However, adding primers at the start gave mostly spurious products; adding primers after 15 cycles gave very strong bands of the correct size. | ||
| + | ** Cleaned up DNA. | ||
| + | ** '''Gibson assembly:''' | ||
| + | *** pET21.BB (control) | ||
| + | *** pET21.BB + rrnB.LasI.RFP.LoxLac.sfGFP.double.terminator | ||
| + | *** pET21.BB + rrnB.LasI.RFP.LoxLacFlipped.sfGFP.double.terminator '''*Note*: These first two rxns used 0.02 pmol total fragments.''' | ||
| + | *** pET21.BB + rrnB.LasI.RFP.LoxLac.sfGFP.double.terminator | ||
| + | *** pET21.BB + rrnB.LasI.RFP.LoxLacFlipped.sfGFP.double.terminator '''*Note*: These last two rxns used 0.20 pmol total fragments.''' | ||
| + | *** '''Results:''' The rxn with 0.20 pmol total fragments gave a product band of the correct size. | ||
| + | ** Desalted, transformed, and plated on LB+Cab. | ||
| + | ** '''Colony PCR:''' '''*Note:* Used far too short elongation time (appropriate for Phusion but not for the Taq master mix I use with colony PCR)''' | ||
| + | *** pReporter: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** '''Results:''' Lots of spurious bands. No correct-sized bands. Will retry with longer elongation time. | ||
| + | ** '''Colony PCR:''' | ||
| + | *** pReporter: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R | ||
| + | *** '''Results:''' Still lots of spurious bands. No correct-sized bands. Will prep and use as template for PCR. | ||
== Spinach Aptamer == | == Spinach Aptamer == | ||
| + | *Logan | ||
| + | **7/16 - Ran PCR reactions on many random colonies from the plate of the construct with mCherry 3' of SpA. | ||
| + | **7/17 - Colony PCR of both 3' and 5' constructs (as in 7/16). Ran GE for PCR products from 7/16 - Found many plasmid with correct insert. | ||
| + | **7/18 - Started cultures at 7:30 AM, came in at 11 and still not mid log phase so continued culturing and will dilute next day. Transformed pAAV-miniCMV-mCherry plasmid into Bl-21 AI cells to use as control in plate reader exp. | ||
| + | **7/19 - Diluted cultures from 7/18 to use in plate reader exp. After dilution, grew 1.5 hours, added 25 uM DFHBI(gfp-like fluorophore) and cultured further 30 mins. Induced with arabinose, and ran SpA, mCherry, and the 5' construct in 96 well plate for 3.5 hours monitoring fluorescent wavelengths of both SpA and mCherry. | ||
| + | **7/20 - Result of plate reader exp: no fluorescence. Did not wait long enough for induction. Pelleted the plate reader cultures from 7/19 (grown with DFHBI and arabinose induced overnight) - Under blue UV box: mCherry - no fluoresence, SpA - little fluorescence, 5' construct - glowing red and green. Transformed 3' construct plasmid in Bl-21 AI, will sequence 7/23. | ||
== CBB5 Caffeine Inducible Promoters == | == CBB5 Caffeine Inducible Promoters == | ||
| + | *Peter | ||
| + | **7/16- Sequencing results failed again. Picked 4 new colonies from ORF1 Gibson plate (ORF1-3,4,5,6) and set to grow overnight in LB-Kan culture. | ||
| + | **7/17- Confirmation PCR of ORF1 -3,4,5,6 cultures. Morning PCR failed, so PCR retried using quickTAQ mastermix overnight. | ||
| + | **7/18- Successful PCR of ORF1 -4,5,6. Miniprepped these 3 cultures, and submitted for sequencing. | ||
| + | **7/20- Successful sequencing of ORF1 -4,5,6. | ||
== Inducible Odor == | == Inducible Odor == | ||
Latest revision as of 21:27, 31 July 2012
Contents |
Notebook for week of July 16 to July 22
Design of Converter Plasmid
- Razan
- 7/16- Tested overlap regions using overlap PCR.
- 7/17- Tested overlap regions 2 pieces at a time using gibson. Ordered new primers to increase backbone overlap region.
Design of Reporter Plasmid
- Ben
- Colony PCR:
- pReporter: JE.rrnB.F, JE.GFPTerm.R
- pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R
- Results: No correctly sized bands. Will retry Gibson assembly with new master mix.
- Gibson assembly:
- pET21.BB (control)
- pET21.BB + rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator
- pET21.BB + rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator
- Results: Still no visible product band. Will retry Gibson assembly after OE-PCRing each set of inserts into 1 piece first.
- OE-PCR:
- rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R
- rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R
- rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R
- rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator: JE.rrnB.F, JE.GFPTerm.R *Note*: These last two rxns did not have primers added until after 15 cycles.
- Results: All lanes contained correct-size bands. However, adding primers at the start gave mostly spurious products; adding primers after 15 cycles gave very strong bands of the correct size.
- Cleaned up DNA.
- Gibson assembly:
- pET21.BB (control)
- pET21.BB + rrnB.LasI.RFP.LoxLac.sfGFP.double.terminator
- pET21.BB + rrnB.LasI.RFP.LoxLacFlipped.sfGFP.double.terminator *Note*: These first two rxns used 0.02 pmol total fragments.
- pET21.BB + rrnB.LasI.RFP.LoxLac.sfGFP.double.terminator
- pET21.BB + rrnB.LasI.RFP.LoxLacFlipped.sfGFP.double.terminator *Note*: These last two rxns used 0.20 pmol total fragments.
- Results: The rxn with 0.20 pmol total fragments gave a product band of the correct size.
- Desalted, transformed, and plated on LB+Cab.
- Colony PCR: *Note:* Used far too short elongation time (appropriate for Phusion but not for the Taq master mix I use with colony PCR)
- pReporter: JE.rrnB.F, JE.GFPTerm.R
- pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R
- Results: Lots of spurious bands. No correct-sized bands. Will retry with longer elongation time.
- Colony PCR:
- pReporter: JE.rrnB.F, JE.GFPTerm.R
- pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R
- Results: Still lots of spurious bands. No correct-sized bands. Will prep and use as template for PCR.
- Colony PCR:
Spinach Aptamer
- Logan
- 7/16 - Ran PCR reactions on many random colonies from the plate of the construct with mCherry 3' of SpA.
- 7/17 - Colony PCR of both 3' and 5' constructs (as in 7/16). Ran GE for PCR products from 7/16 - Found many plasmid with correct insert.
- 7/18 - Started cultures at 7:30 AM, came in at 11 and still not mid log phase so continued culturing and will dilute next day. Transformed pAAV-miniCMV-mCherry plasmid into Bl-21 AI cells to use as control in plate reader exp.
- 7/19 - Diluted cultures from 7/18 to use in plate reader exp. After dilution, grew 1.5 hours, added 25 uM DFHBI(gfp-like fluorophore) and cultured further 30 mins. Induced with arabinose, and ran SpA, mCherry, and the 5' construct in 96 well plate for 3.5 hours monitoring fluorescent wavelengths of both SpA and mCherry.
- 7/20 - Result of plate reader exp: no fluorescence. Did not wait long enough for induction. Pelleted the plate reader cultures from 7/19 (grown with DFHBI and arabinose induced overnight) - Under blue UV box: mCherry - no fluoresence, SpA - little fluorescence, 5' construct - glowing red and green. Transformed 3' construct plasmid in Bl-21 AI, will sequence 7/23.
CBB5 Caffeine Inducible Promoters
- Peter
- 7/16- Sequencing results failed again. Picked 4 new colonies from ORF1 Gibson plate (ORF1-3,4,5,6) and set to grow overnight in LB-Kan culture.
- 7/17- Confirmation PCR of ORF1 -3,4,5,6 cultures. Morning PCR failed, so PCR retried using quickTAQ mastermix overnight.
- 7/18- Successful PCR of ORF1 -4,5,6. Miniprepped these 3 cultures, and submitted for sequencing.
- 7/20- Successful sequencing of ORF1 -4,5,6.
