GcatWiki - User contributions [en]

Electroporation Transformation

2014-07-10T19:40:06Z

Ekeffeler:

'''This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA'''

'''Cell Preparation'''

1. This procedure yields enough bacteria for two transformations - scale up for more

2. Grow bacteria to be transformed in 40 ml of broth (eg. LB Broth) in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight)

3. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH2O

4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O

5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O

6. Transfer to a sterile 1.5 mL tube and pellet the culture by centrifuging in a microfuge for 1 minute; resuspend in 80 ul sterile 10% glycerol

'''Electroporation'''

1. Use a clean electroporation cuvette with chamber gap of 1 mm and set charging voltage to 1.3 - 1.5 kV

2. Pipette 1 ul of plasmid (1 ng/ul) into the electroporation cuvette

3. Add 40 ul of electrocompetent bacteria to the cuvette and flick gently

4. Click the Pulse button

5. Transfer to sterile 1.5 ml tube and immediately add 960 ul sterile LB or SOC

6. Incubate at 37 C with shaking for 1 hour

7. Spin in microfuge for 60 seconds, pour off all supernatant

8. Add 50 ul LB + antibiotic, resuspend pellet, and spread onto agar plate with antibiotic

'''Washing Electroporation Cuvettes'''

1. Fill the cuvettes two times with dH2O, dumping each time

2. Fill the cuvettes with 0.1 N NaOH and let sit for 5 minutes

3. Fill the cuvettes two times with dH2O, dumping each time

4. Fill the cuvettes two times with 95% ethanol, dumping each time

5. Turn upside down to dry

LongAmp PCR

2014-07-10T18:55:36Z

Ekeffeler:

== Preparation ==

1. 0.6uL 10mM dNTPs

2. 1uL 1ng template

3. 1uL 10uM forward primer

4. 1uL 10uM reverse primer

5. 1uL LongAmp Taq Polymerase

6. 4uL 5X LongAmp Buffer

7. 11.4uL dH20

== Programed PCR Protocol ==

'''Stage 1:'''

94ºC 5 minutes

'''Stage 2:'''

30 cycles

94ºC 30 seconds

55ºC 30 seconds (Or a different annealing temperature)

65ºC 10 minutes (Or 1 minute per 1000bp)

'''Stage 3:'''

65ºC 10 minutes

MWSU protocols

2014-07-01T18:33:51Z

Ekeffeler:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

[[Template Preparation for RT-qPCR]]

[[New Chaperone PCR]]

[[LongAmp PCR]]

'''Recombinant DNA Production'''

[[Zymo Research Plasmid Minipreps]]

[[Golden Gate Assembly Protocol for BsmB1]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

[[File: PClone_Procedure_for_GCAT_SB_Workshop_2014_new_version.pptx]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Electroporation Transformation]]

[[Bacterial Media]]

[[Washing Beads]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

LongAmp PCR

2014-07-01T18:32:42Z

Ekeffeler: Created page with "== Preparation == 1. 0.6uL 10mM dNTPs 2. 1uL 1ng template 3. 1uL 10uM forward primer 4. 1uL 10uM reverse primer 5. 1uL LongAmp Taq Polymerase 6. 4uL LongAmp Buffer 7. 1..."

== Preparation ==

1. 0.6uL 10mM dNTPs

2. 1uL 1ng template

3. 1uL 10uM forward primer

4. 1uL 10uM reverse primer

5. 1uL LongAmp Taq Polymerase

6. 4uL LongAmp Buffer

7. 11.4uL dH20

== Programed PCR Protocol ==

'''Stage 1:'''

94ºC 5 minutes

'''Stage 2:'''

30 cycles

94ºC 30 seconds

60ºC-->45ºC 30 seconds

60ºC 10 minutes

'''Stage 3:'''

72ºC 10 minutes

MWSU protocols

2014-07-01T18:26:06Z

Ekeffeler:

'''Purification of DNA'''
[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

[[Template Preparation for RT-qPCR]]

[[New Chaperone PCR]]

[[LongAmp PCR]]

'''Recombinant DNA Production'''

[[Zymo Research Plasmid Minipreps]]

[[Golden Gate Assembly Protocol for BsmB1]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

[[File: PClone_Procedure_for_GCAT_SB_Workshop_2014_new_version.pptx]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Electroporation Transformation]]

[[Bacterial Media]]

[[Washing Beads]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

File:Hans Peter Sørensen.pdf

2014-06-18T20:57:20Z

Ekeffeler:

References

2014-06-18T20:56:56Z

Ekeffeler:

Applications of Genetically-Encoded Biosensors for the
Construction and Control of Biosynthetic Pathways [[File:Josh K. Michener.pdf]]

Synthetic RNA switches as a tool for temporal and spatial
control over gene expression [[File:Andrew L. Chang.pdf]]

Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein [[File:F. Ulrich Hartl.pdf]]

Soluable expression of recombinant proteins in the cytoplasm of E.coli [[File:Hans Peter Sørensen.pdf]]

File:F. Ulrich Hartl.pdf

2014-06-18T20:31:20Z

Ekeffeler:

References

2014-06-18T20:31:05Z

Ekeffeler:

File:Andrew L. Chang.pdf

2014-06-18T14:52:51Z

Ekeffeler: Ekeffeler uploaded a new version of "File:Andrew L. Chang.pdf"

File:Josh K. Michener.pdf

2014-06-18T14:52:19Z

Ekeffeler: Ekeffeler uploaded a new version of "File:Josh K. Michener.pdf"

File:Andrew L. Chang.pdf

2014-06-17T21:11:26Z

Ekeffeler:

References

2014-06-17T21:11:14Z

Ekeffeler:

File:Josh K. Michener.pdf

2014-06-17T21:09:24Z

Ekeffeler:

References

2014-06-17T21:08:58Z

Ekeffeler:

Applications of Genetically-Encoded Biosensors for the
Construction and Control of Biosynthetic Pathways [[File:Josh K. Michener.pdf]]

References

2014-06-17T21:08:44Z

Ekeffeler:

Applications of Genetically-Encoded Biosensors for the
Construction and Control of Biosynthetic Pathways [[File:Josh K. Michener.pdf]]

References

2014-06-17T16:35:02Z

Ekeffeler: Blanked the page

Summer 2014 SynBio Project (Davidson and MWSU)

2014-06-17T16:34:46Z

Ekeffeler:

=== Presentations during MWSU visit to Davidson ===

Central Dogma presentation [[File:Central_dogma.pptx]]

PCR presentation [[File:PCR.pptx]]

Cloning presentation [[File:Cloning.pptx]]

Riboswitch presentation [[File:Riboswitch_function.pptx]]

Agent based modeling presentation [[File:AgentBased_Modeling.pptx]]

Competition presentation [[File:Competition_Modeling.xlsx]]

Programmed evolution presentation [[File:Programmed_Evolution.pdf]]

Caffeine results [[File:Caffeine_Disk.pptx]]

Ammeline presentation [[File:Ammeline.pptx]]

Repressilator modeling exercises [[File:repressilator_modeling.docx]]

Repressilator modeling Excel file [[File:Repressilator_model.xls]]

Repressilator modeling Netlogo file [[File:Repressilator_mod.nlogo.zip]]

=== Biology Files ===

Chaperone plasmid DNA sequences [[File:Chaperone_plasmid_DNA_sequences.docx]]

Origin PCR Gels 5-26-14[[File:5-26-14 Orig PCR Elecpho Gel Pic.doc]]

Chaperone PCR and Repeat Origin PCR 5-27-14[[File:5-27-14_Chap_PCR_and_Repeat_Ori_PCR.doc]]

Chaperone PCR of original clones 5-27-14[[File:5-27-14_chaperone_pcr_clones_1-24.doc]]

Origin PCR of original clones 5-27-14[[File:5-27-14 Ori PCR 1-24.doc ]]

New Chap PCR clones 1-5[[File:5-28-14_New_Chap_PCR_1-5.doc ]]

Combinations Labeling System [[File:Labeling.xlsx]]

ThyA Fitness Module [[File:ThyA fitness module.docx]]

Alternative Riboswitches[[File:Alternative Riboswitches.docx]]

Origin PCR Pictures 6-5-14 [[File:6-5-14 Origin PCR.docx]]

Chaperone PCR PA 6-5-14 [[File:6-5-14 Chaperone PCR.docx]]

Broth Growth Experiments [[File:Broth Growth Experiment Reports Revised.docx]]
===Sub-pages on various topics===

'''
[[MATH]]
'''

[[Catherine Doyle Thesis Materials]]

[[Repeating 20 Clone Experiments]]

[[Extending theophylline application]]

[[Melamine iteration]]

[[Ramping up Programmed Evolution]]

[[Rational Design of Riboswitches: papers to read]]

[[ThyA Fitness Module]]

[[Caffeine Disk Replication Data]]

=== Programmed Evolution Paper ===

[[References]]

Summer 2014 SynBio Project (Davidson and MWSU)

2014-06-17T16:33:55Z

Ekeffeler:

Additional Ideas for Future Programmed Evolution Research

2014-06-11T21:34:20Z

Ekeffeler:

1. New System-

New Riboswitch, Multistep

-Ex: Adenosylcobalamin riboswitch-
This is possibly a good multi-step pathway, but E. coli metabolism may be too centered on the cobalamin system, making it a concern to disrupt the system and not a good option for further research. A system similar to this multi-step pathway would be a great option, as long as the pathway would not disrupt metabolism and have an ill-effect on the survival and regrowth of the bacteria. During discussion, both ideas for a new system were related by being in the vitamin family, due to the articles found while researching. So this may be an indicator to take a more heterologous approach while searching for a new system and riboswitch that would be more foreign and non-dependent for the E. coli metabolism.

-Ex:Thiamine pyrophosphate riboswitch [[File:Thiamine_Pyrophosphate_Riboswitch.pdf]]

In E.coli, thiamine monophosphate is converted to thiamine which then could be converted to thiamine pyrophosphate by thiamine kinase. For this particular riboswitch when thiamine pyrophosphate (TPP) binds to the riboswitch, it will stop transcription. A discussion about the thiamine pyrophosphate riboswitch resulted in the decision to not pursue this specific riboswitch because E.coli relies on this metabolic system to survive and it could be impossible to remove thiamine and still have them survive. This riboswitch is a great example of a multi-step metabolic pathway, but unfortunately it would be too detrimental to remove thiamine from E.coli’s system. It is still a goal to research a multi-step pathway as fascinating as the thiamine->>thiamine pyrophosphate, but still remain similar and practical to the caffeine->theophylline system.

2. Biosynthesis of Caffeine then Theophylline-
•Making our single-step pathway to a multi-step pathway

3. Manipulating the promoter, RBS, origin, and chaperone-
•Ex: 5 promoters and 5 RBS’s or 500 promoters and 500 RBS’s

4. A. Measure clone distribution around different caffeine concentrations (disks)-
•Coloring the clock
4. B. Measure clone distribution on different caffeine concentrations (plates)-
•Ex: 2mM Caffeine -> clone #23 or 4mM caffeine-> clone ?
•Cranking up selection

Additional Ideas for Future Programmed Evolution Research

2014-06-11T21:31:16Z

Ekeffeler:

1. New System-

New Riboswitch, Multistep

-Ex: Adenosylcobalamin riboswitch-
This is possibly a good multi-step pathway, but E. coli metabolism may be too centered on the cobalamin system, making it a concern to disrupt the system and not a good option for further research. A system similar to this multi-step pathway would be a great option, as long as the pathway would not disrupt metabolism and have an ill-effect on the survival and regrowth of the bacteria. During discussion, both ideas for a new system were related by being in the vitamin family, due to the articles found while researching. So this may be an indicator to take a more heterologous approach while searching for a new system and riboswitch that would be more foreign and non-dependent for the E. coli metabolism.

-Ex:Thiamine pyrophosphate riboswitch [[File:Thiamine_Pyrophosphate_Riboswitch.pdf]]

In E.coli, thiamine monophosphate is converted to thiamine which then could be converted to thiamine pyrophosphate by thiamine kinase. For this particular riboswitch when thiamine pyrophosphate (TPP) binds to the riboswitch, it will stop transcription. A discussion about the thiamine pyrophosphate riboswitch resulted in the decision to not pursue this specific riboswitch because E.coli relies on this metabolic system to survive and it could be impossible to remove thiamine and still have them survive. This riboswitch is a great example of a multi-step metabolic pathway, but unfortunately it would be too detrimental to remove thiamine from E.coli’s system. It is still a goal to research a multi-step pathway as fascinating as the thiamine->>thiamine pyrophosphate, but still remain similar and practical to the caffeine->theophylline system.

2. Biosynthesis of Caffeine then Theophylline-
•Making our single-step pathway to a multi-step pathway

3. Manipulating the promoter, RBS, origin, and chaperone-
•Ex: 5 promoters and 5 RBS’s or 500 promoters and 500 RBS’s

4. A. Measure clone distribution around different caffeine concentrations (disks)-
•Coloring the clock
B. Measure clone distribution on different caffeine concentrations (plates)-
•Ex: 2mM Caffeine -> clone #23 or 4mM caffeine-> clone ?
•Cranking up selection

Additional Ideas for Future Programmed Evolution Research

2014-06-11T19:35:40Z

Ekeffeler:

1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)

2. Thiamine pyrophosphate riboswitch [[File:Thiamine_Pyrophosphate_Riboswitch.pdf]]

-In E.coli, thiamine monophosphate is converted to thiamine which then could be converted to thiamine pyrophosphate by thiamine kinase. For this particular riboswitch when thiamine pyrophosphate (TPP) binds to the riboswitch, it will stop transcription. A discussion about the thiamine pyrophosphate riboswitch resulted in the decision to not pursue this specific riboswitch because E.coli relies on this metabolic system to survive and it could be impossible to remove thiamine and still have them survive. This riboswitch is a great example of a multi-step metabolic pathway, but unfortunately it would be too detrimental to remove thiamine from E.coli’s system. It is still a goal to research a multi-step pathway as fascinating as the thiamine->>thiamine pyrophosphate, but still remain similar and practical to the caffeine->theophylline system.

Additional Ideas for Future Programmed Evolution Research

2014-06-11T19:35:16Z

Ekeffeler:

Additional Ideas for Future Programmed Evolution Research

2014-06-11T19:35:00Z

Ekeffeler:

1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)

2. Thiamine pyrophosphate riboswitch [[File:Thiamine_Pyrophosphate_Riboswitch.pdf]]

--In E.coli, thiamine monophosphate is converted to thiamine which then could be converted to thiamine pyrophosphate by thiamine kinase. For this particular riboswitch when thiamine pyrophosphate (TPP) binds to the riboswitch, it will stop transcription. A discussion about the thiamine pyrophosphate riboswitch resulted in the decision to not pursue this specific riboswitch because E.coli relies on this metabolic system to survive and it could be impossible to remove thiamine and still have them survive. This riboswitch is a great example of a multi-step metabolic pathway, but unfortunately it would be too detrimental to remove thiamine from E.coli’s system. It is still a goal to research a multi-step pathway as fascinating as the thiamine->>thiamine pyrophosphate, but still remain similar and practical to the caffeine->theophylline system.

Additional Ideas for Future Programmed Evolution Research

2014-06-11T16:01:19Z

Ekeffeler:

1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)

2. Thiamine riboswitch [[File:Thiamine_Pyrophosphate_Riboswitch.pdf]]

Additional Ideas for Future Programmed Evolution Research

2014-06-11T15:59:14Z

Ekeffeler:

1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)

2. Thiamine riboswitch [[Thiamine_Pyrophosphate_Riboswitch.pdf]]

File:Thiamine Pyrophosphate Riboswitch.pdf

2014-06-11T15:57:46Z

Ekeffeler:

Additional Ideas for Future Programmed Evolution Research

2014-06-11T15:56:47Z

Ekeffeler:

1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)

2. Thiamine riboswitch

Additional Ideas for Future Programmed Evolution Research

2014-06-11T15:47:06Z

Ekeffeler: Created page with '1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)'

1. Develop a multistep pathway off of the caffeine->theophylline single step pathway (Find a substrate that creates caffeine)

Ramping up Programmed Evolution

2014-06-11T15:45:26Z

Ekeffeler:

1) Riboswitch
* Dual ligand
* Turning on and off riboswitch
2) Increased variations in the module including the chaperones
* Increase in variation within current categories. (Copy number, promoter strength)
* New category: mutation of individual base pairs
* Controlling interaction of riboswitch and aptamer
3) Ecoli-yeast
* Temperature difference
* Simple vs. complex pathway of translation
4) The number of steps in the metabolic pathway
* The formation of byproducts
* More than one compound of interest, possibly by decomposition.
* The number of steps to reach differences in significant results.
5) Introducing mutations like in the promoter region, the ribosome binding site, elements found in nature like nickel, cadmium
6) Utilize other bacterial species i.e. B.subtilius

[[Team 1's Brainstorm for Future Programmed Evolution Research]]

[[Additional Ideas for Future Programmed Evolution Research]]

File:MWSU3.xlsx

2014-06-11T15:41:36Z

Ekeffeler:

File:MWSU2.pptx

2014-06-11T15:41:23Z

Ekeffeler:

Caffeine Disk Replication Data

2014-06-11T15:41:06Z

Ekeffeler:

MWSU Caffeine Disk Replication PowerPoint [[File:MWSU.pptx]]

MWSU Caffeine Disk Replication Excel Spreadsheet [[File:MWSU.xlsx]]

MWSU Trial Run Caffeine Disk Replication PowerPoint [[File:MWSU2.pptx]]

MWSU Trial Run Caffeine Disk Replication Excel Spreadsheet [[File:MWSU3.xlsx]]

Caffeine Disk Replication Data

2014-06-11T15:39:39Z

Ekeffeler:

Caffeine Disk Replication Data

2014-06-11T15:38:53Z

Ekeffeler:

MWSU Caffeine Disk Replication PowerPoint [[File:MWSU.pptx]]

MWSU Caffeine Disk Replication Excel Spreadsheet [[File:MWSU.xlsx]]

MWSU Trial Run Caffeine Disk Replication PowerPoint [[File:MWSU1.pptx]]

MWSU Trial Run Caffeine Disk Replication Excel Spreadsheet [[File:MWSU2.xlsx]]

File:MWSU.xlsx

2014-06-11T15:36:30Z

Ekeffeler:

File:MWSU.pptx

2014-06-11T15:34:59Z

Ekeffeler:

File:MWSU1.pptx

2014-06-11T15:34:15Z

Ekeffeler: uploaded a new version of "File:MWSU1.pptx"

Caffeine Disk Replication Data

2014-06-11T15:33:43Z

Ekeffeler:

MWSU Caffeine Disk Replication PowerPoint [[File:MWSU.pptx]]

MWSU Caffeine Disk Replication Excel Spreadsheet [[File:MWSU.xlsx]]

Caffeine Disk Replication Data

2014-06-11T15:33:21Z

Ekeffeler:

MWSU Caffeine Disk Replication PowerPoint [[File:MWSU1.pptx]]

MWSU Caffeine Disk Replication Excel Spreadsheet [[File:MWSU2.xlsx]]

Caffeine Disk Replication Data

2014-06-10T17:40:59Z

Ekeffeler: Blanked the page

File:MWSU2.xlsx

2014-06-10T17:37:12Z

Ekeffeler:

Caffeine Disk Replication Data

2014-06-10T17:36:58Z

Ekeffeler:

MWSU Caffeine Disk Replication PowerPoint [[File:MWSU1.pptx]]

MWSU Caffeine Disk Replication Excel Spreadsheet [[File:MWSU2.xlsx]]

File:MWSU1.pptx

2014-06-10T17:02:54Z

Ekeffeler:

File:MWSU Caffeine disk replication data 6-10-14 (1).pptx

2014-06-10T17:00:48Z

Ekeffeler:

Caffeine Disk Replication Data

2014-06-10T16:56:26Z

Ekeffeler: Created page with 'MWSU Caffeine Disk Replication PowerPoint File:MWSU1.pptx'

MWSU Caffeine Disk Replication PowerPoint [[File:MWSU1.pptx]]

Summer 2014 SynBio Project (Davidson and MWSU)

2014-06-10T16:54:23Z

Ekeffeler:

=== Presentations during MWSU visit to Davidson ===

Central Dogma presentation [[File:Central_dogma.pptx]]

PCR presentation [[File:PCR.pptx]]

Cloning presentation [[File:Cloning.pptx]]

Riboswitch presentation [[File:Riboswitch_function.pptx]]

Agent based modeling presentation [[File:AgentBased_Modeling.pptx]]

Competition presentation [[File:Competition_Modeling.xlsx]]

Programmed evolution presentation [[File:Programmed_Evolution.pdf]]

Caffeine results [[File:Caffeine_Disk.pptx]]

Ammeline presentation [[File:Ammeline.pptx]]

Repressilator modeling exercises [[File:repressilator_modeling.docx]]

Repressilator modeling Excel file [[File:Repressilator_model.xls]]

Repressilator modeling Netlogo file [[File:Repressilator_mod.nlogo.zip]]

=== Biology Files ===

Chaperone plasmid DNA sequences [[File:Chaperone_plasmid_DNA_sequences.docx]]

Origin PCR Gels 5-26-14[[File:5-26-14 Orig PCR Elecpho Gel Pic.doc]]

Chaperone PCR and Repeat Origin PCR 5-27-14[[File:5-27-14_Chap_PCR_and_Repeat_Ori_PCR.doc]]

Chaperone PCR of original clones 5-27-14[[File:5-27-14_chaperone_pcr_clones_1-24.doc]]

Origin PCR of original clones 5-27-14[[File:5-27-14 Ori PCR 1-24.doc ]]

New Chap PCR clones 1-5[[File:5-28-14_New_Chap_PCR_1-5.doc ]]

Combinations Labeling System [[File:Labeling.xlsx]]

ThyA Fitness Module [[File:ThyA fitness module.docx]]

Alternative Riboswitches[[File:Alternative Riboswitches.docx]]

Origin PCR Pictures 6-5-14 [[File:6-5-14 Origin PCR.docx]]

Chaperone PCR PA 6-5-14 [[File:6-5-14 Chaperone PCR.docx]]
===Sub-pages on various topics===

'''
[[MATH]]
'''

[[Catherine Doyle Thesis Materials]]

[[Repeating 20 Clone Experiments]]

[[Extending theophylline application]]

[[Melamine iteration]]

[[Ramping up Programmed Evolution]]

[[Rational Design of Riboswitches: papers to read]]

[[ThyA Fitness Module]]

[[Caffeine Disk Replication Data]]

File:5-27-14 Ori PCR 1-24.doc

2014-05-29T17:05:31Z

Ekeffeler: uploaded a new version of "File:5-27-14 Ori PCR 1-24.doc"

Origin PCR of original stock of clones 1-24

File:5-26-14 Orig PCR Elecpho Gel Pic.doc

2014-05-29T17:04:21Z

Ekeffeler: uploaded a new version of "File:5-26-14 Orig PCR Elecpho Gel Pic.doc"

Origin PCR Gel 5-26-14

New Chaperone PCR

2014-05-28T19:10:08Z

Ekeffeler:

== Preparation ==

1. 2uL Bacteria

2. 0.5uL New Chaperone Mix

3. 7.5uL dH20

4. 10uL GoTac Green

== Programed PCR Protocol ==

'''Stage 1:'''

94ºC 10 minutes

'''Stage 2:'''

20 cycles

94ºC 15 seconds

51ºC 15 seconds

74ºC 2 minutes

'''Stage 3:'''

74ºC 5 minutes