GcatWiki - User contributions [en]

Template Preparation for RT-qPCR

2013-07-30T16:33:56Z

Bgrieshaber:

'''Template Preparation'''

1. Place 1mL of grown bacteria into a 1.5mL tube.

2. Heat the tube at 95C for 10min in a water bath.

3. Immediately after heating, place the tubes in a -20C cooler/freezer for 40-60min or until frozen.

4. Take out and allow to thaw for dilutions.

'''Dilutions of Prepared Templates'''

After prepared templates have thawed perform serial dilutions of 10^2, 10^2.5, 10^3, 10^3.5, 10^4 making sure to vortex before each dilution.

Template Preparation for RT-qPCR

2013-06-28T17:14:15Z

Bgrieshaber: Created page with "'''Template Preparation''' 1. Place 1mL of grown bacteria into a 1.5mL tube. 2. Heat the tube at 95C for 5min in a water bath. (If tubes pop open frequently try reducing the..."

'''Template Preparation'''

1. Place 1mL of grown bacteria into a 1.5mL tube.

2. Heat the tube at 95C for 5min in a water bath. (If tubes pop open frequently try reducing the volume to .5mL, and/or reinforce lids with tape or clip.)

3. Immediately after heating, place the tubes in a -20C cooler/freezer for 10min or until frozen.

4. Take out and allow to thaw for dilutions.

'''Dilutions of Prepared Templates'''

After prepared templates have thawed perform serial dilutions of 1:10, 1:100, 1:1000 making sure to vortex before each dilution.

-When using 1mL volume of template, dilute .1mL of template into .9mL of dH2O.

-When using .5mL volume of template, dilute .05mL of template into .45mL of dH2O.

MWSU protocols

2013-06-28T16:52:48Z

Bgrieshaber:

'''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]]

'''Recombinant DNA Production'''

[[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]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Bacterial Media]]

'''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]]

Origin of Replication

2013-06-17T15:04:14Z

Bgrieshaber:

'''RNA I and RNA II gene and promoter sites in pSB1a2-BR'''

[[File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎]]

This is an link to the document containing the sequences for RNA I and RNA II sites as well as their promoters. The document contains annotations as to the location of each element. *Note that the full length of RNA II is not fully known but is a close approximation.

----
'''RNA I/RNA II Literature'''

Replication and Control of Circular Bacterial Plasmids
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98921/pdf/mr000434.pdf]

Plasmid ColE1 Incompatibility Determined by Interaction of RNA I
with Primer Transcript
[http://www.pnas.org/content/78/10/6096.full.pdf]

Formation of an RNA Primer for Initiation of Replication of ColE1
DNA by ribonuclease H
[http://www.pnas.org/content/77/5/2450.full.pdf]

Control of pMB1 Replication: Inhibition of Primer Formation
by Rop Requires RNA1
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557524/pdf/emboj00310-0150.pdf]

High Copy Number of the pUC Plasmid Results from a Rom/Rop--Suppressible Point Mutation in RNA II.
[http://content.ebscohost.com/pdf9/pdf/1992/ESL/15Nov92/15903115.pdf?T=P&P=AN&K=15903115&S=R&D=eih&EbscoContent=dGJyMMTo50SeprU4yOvsOLCmr0uep65Ssa24SLSWxWXS&ContentCustomer=dGJyMK6nr0W2pq5T69fnhrnb5ofx6gAA]

Control of ColE1 Replication: Low Affinity Specific Binding
of Rop (Rom) to RNAI and RNAII
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC454354/pdf/emboj00139-0256.pdf]

Plasmid pT181 Replication is Regulated by Two Countertranscripts
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397100/pdf/pnas00343-0012.pdf]

The mutH Gene Regulates the Replication and Methylation
of the pMB1 Origin
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207916/pdf/jbacter00100-0187.pdf]

Attempt to Construct a Rop+ pUC19 from pBR322 Mutant Lacking
the Tetracycline Resistance Gene
[http://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/10/10-18.pdf]

'''Quantitative PCR Literature'''

Improved Determination of Plasmid Copy Number Using Quantitative Real-Time PCR for Monitoring Fermentation Processes
[http://www.microbialcellfactories.com/content/7/1/6]

Fast SYBR Green Master Mix Protocol
[http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_046776.pdf]
----
'''[[Oligos and Primers]]'''

Oligos and Primers

2013-06-17T13:57:15Z

Bgrieshaber:

Oligo Designs for Mutagenesis of RNA1 promoter and +1 region:
[[File:RNA 1 J-GGA oligos 6-6-13 Final.docx]]

Primer Design for Measuring PCN with Real Time PCR
[[File:Primer Design for Measuring PCN with Real Time PCR.docx]]

Oligos and Primers

2013-06-17T13:56:02Z

Bgrieshaber:

Oligo Designs for Mutagenesis of RNA1 promoter and +1 region:
[[File:RNA 1 J-GGA oligos 6-6-13 Final.docx]]

Primer Design for Measuring PCN with Real Time PCR
[[File:http://gcat.davidson.edu/mediawiki-1.19.1/images/a/a1/Primer_Design_for_Measuring_PCN_with_Real_Time_PCR.docx]]

File:Primer Design for Measuring PCN with Real Time PCR.docx

2013-06-17T13:53:36Z

Bgrieshaber:

Origin of Replication

2013-06-11T21:42:22Z

Bgrieshaber:

Origin of Replication

2013-06-10T18:17:19Z

Bgrieshaber:

'''RNA I and RNA II gene and promoter sites in pSB1a2-BR'''

[[File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎]]

This is an link to the document containing the sequences for RNA I and RNA II sites as well as their promoters. The document contains annotations as to the location of each element. *Note that the full length of RNA II is not fully known but is a close approximation.

----
'''Source Literature'''

Replication and Control of Circular Bacterial Plasmids
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98921/pdf/mr000434.pdf]

Plasmid ColE1 Incompatibility Determined by Interaction of RNA I
with Primer Transcript
[http://www.pnas.org/content/78/10/6096.full.pdf]

Formation of an RNA Primer for Initiation of Replication of ColE1
DNA by ribonuclease H
[http://www.pnas.org/content/77/5/2450.full.pdf]

Control of pMB1 Replication: Inhibition of Primer Formation
by Rop Requires RNA1
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557524/pdf/emboj00310-0150.pdf]

High Copy Number of the pUC Plasmid Results from a Rom/Rop--Suppressible Point Mutation in RNA II.
[http://content.ebscohost.com/pdf9/pdf/1992/ESL/15Nov92/15903115.pdf?T=P&P=AN&K=15903115&S=R&D=eih&EbscoContent=dGJyMMTo50SeprU4yOvsOLCmr0uep65Ssa24SLSWxWXS&ContentCustomer=dGJyMK6nr0W2pq5T69fnhrnb5ofx6gAA]

Control of ColE1 Replication: Low Affinity Specific Binding
of Rop (Rom) to RNAI and RNAII
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC454354/pdf/emboj00139-0256.pdf]

Plasmid pT181 Replication is Regulated by Two Countertranscripts
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397100/pdf/pnas00343-0012.pdf]

The mutH Gene Regulates the Replication and Methylation
of the pMB1 Origin
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207916/pdf/jbacter00100-0187.pdf]

Attempt to Construct a Rop+ pUC19 from pBR322 Mutant Lacking
the Tetracycline Resistance Gene
[http://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/10/10-18.pdf]

----
'''[[Oligos and Primers]]'''

Oligos and Primers

2013-06-10T18:16:02Z

Bgrieshaber:

Oligo Designs for Mutagenesis of RNA1 promoter and +1 region:
[[File:RNA 1 J-GGA oligos 6-6-13 Final.docx]]

Oligos and Primers

2013-06-10T18:15:35Z

Bgrieshaber: Created page with "'''Oligo Designs for Mutagenesis of RNA1 promoter and +1 region:''' File:RNA 1 J-GGA oligos 6-6-13 Final.docx"

'''Oligo Designs for Mutagenesis of RNA1 promoter and +1 region:'''
[[File:RNA 1 J-GGA oligos 6-6-13 Final.docx]]

File:RNA 1 J-GGA oligos 6-6-13 Final.docx

2013-06-10T18:12:52Z

Bgrieshaber:

Origin of Replication

2013-06-10T17:52:29Z

Bgrieshaber:

'''RNA I and RNA II gene and promoter sites in pSB1a2-BR'''

[[File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎]]

This is an link to the document containing the sequences for RNA I and RNA II sites as well as their promoters. The document contains annotations as to the location of each element. *Note that the full length of RNA II is not fully known but is a close approximation.

----
'''Source Literature'''

Replication and Control of Circular Bacterial Plasmids
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98921/pdf/mr000434.pdf]

Plasmid ColE1 Incompatibility Determined by Interaction of RNA I
with Primer Transcript
[http://www.pnas.org/content/78/10/6096.full.pdf]

Formation of an RNA Primer for Initiation of Replication of ColE1
DNA by ribonuclease H
[http://www.pnas.org/content/77/5/2450.full.pdf]

Control of pMB1 Replication: Inhibition of Primer Formation
by Rop Requires RNA1
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557524/pdf/emboj00310-0150.pdf]

High Copy Number of the pUC Plasmid Results from a Rom/Rop--Suppressible Point Mutation in RNA II.
[http://content.ebscohost.com/pdf9/pdf/1992/ESL/15Nov92/15903115.pdf?T=P&P=AN&K=15903115&S=R&D=eih&EbscoContent=dGJyMMTo50SeprU4yOvsOLCmr0uep65Ssa24SLSWxWXS&ContentCustomer=dGJyMK6nr0W2pq5T69fnhrnb5ofx6gAA]

Control of ColE1 Replication: Low Affinity Specific Binding
of Rop (Rom) to RNAI and RNAII
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC454354/pdf/emboj00139-0256.pdf]

Plasmid pT181 Replication is Regulated by Two Countertranscripts
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397100/pdf/pnas00343-0012.pdf]

The mutH Gene Regulates the Replication and Methylation
of the pMB1 Origin
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207916/pdf/jbacter00100-0187.pdf]

Attempt to Construct a Rop+ pUC19 from pBR322 Mutant Lacking
the Tetracycline Resistance Gene
[http://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/10/10-18.pdf]

----
[[Oligos and Primers]]

Origin of Replication

2013-06-10T17:52:16Z

Bgrieshaber:

'''RNA I and RNA II gene and promoter sites in pSB1a2-BR'''

[[File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎]]

This is an link to the document containing the sequences for RNA I and RNA II sites as well as their promoters. The document contains annotations as to the location of each element. *Note that the full length of RNA II is not fully known but is a close approximation.

----
'''Source Literature'''

Replication and Control of Circular Bacterial Plasmids
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98921/pdf/mr000434.pdf]

Plasmid ColE1 Incompatibility Determined by Interaction of RNA I
with Primer Transcript
[http://www.pnas.org/content/78/10/6096.full.pdf]

Formation of an RNA Primer for Initiation of Replication of ColE1
DNA by ribonuclease H
[http://www.pnas.org/content/77/5/2450.full.pdf]

Control of pMB1 Replication: Inhibition of Primer Formation
by Rop Requires RNA1
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557524/pdf/emboj00310-0150.pdf]

High Copy Number of the pUC Plasmid Results from a Rom/Rop--Suppressible Point Mutation in RNA II.
[http://content.ebscohost.com/pdf9/pdf/1992/ESL/15Nov92/15903115.pdf?T=P&P=AN&K=15903115&S=R&D=eih&EbscoContent=dGJyMMTo50SeprU4yOvsOLCmr0uep65Ssa24SLSWxWXS&ContentCustomer=dGJyMK6nr0W2pq5T69fnhrnb5ofx6gAA]

Control of ColE1 Replication: Low Affinity Specific Binding
of Rop (Rom) to RNAI and RNAII
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC454354/pdf/emboj00139-0256.pdf]

Plasmid pT181 Replication is Regulated by Two Countertranscripts
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397100/pdf/pnas00343-0012.pdf]

The mutH Gene Regulates the Replication and Methylation
of the pMB1 Origin
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207916/pdf/jbacter00100-0187.pdf]

Attempt to Construct a Rop+ pUC19 from pBR322 Mutant Lacking
the Tetracycline Resistance Gene
[http://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/10/10-18.pdf]

----
[[Oligos and Primers]]

Origin of Replication

2013-06-07T14:49:21Z

Bgrieshaber:

'''RNA I and RNA II gene and promoter sites in pSB1a2-BR'''

[[File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎]]

This is an link to the document containing the sequences for RNA I and RNA II sites as well as their promoters. The document contains annotations as to the location of each element. *Note that the full length of RNA II is not fully known but is a close approximation.

----
'''Source Literature'''

Replication and Control of Circular Bacterial Plasmids
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98921/pdf/mr000434.pdf]

Plasmid ColE1 Incompatibility Determined by Interaction of RNA I
with Primer Transcript
[http://www.pnas.org/content/78/10/6096.full.pdf]

Formation of an RNA Primer for Initiation of Replication of ColE1
DNA by ribonuclease H
[http://www.pnas.org/content/77/5/2450.full.pdf]

Control of pMB1 Replication: Inhibition of Primer Formation
by Rop Requires RNA1
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557524/pdf/emboj00310-0150.pdf]

High Copy Number of the pUC Plasmid Results from a Rom/Rop--Suppressible Point Mutation in RNA II.
[http://content.ebscohost.com/pdf9/pdf/1992/ESL/15Nov92/15903115.pdf?T=P&P=AN&K=15903115&S=R&D=eih&EbscoContent=dGJyMMTo50SeprU4yOvsOLCmr0uep65Ssa24SLSWxWXS&ContentCustomer=dGJyMK6nr0W2pq5T69fnhrnb5ofx6gAA]

Control of ColE1 Replication: Low Affinity Specific Binding
of Rop (Rom) to RNAI and RNAII
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC454354/pdf/emboj00139-0256.pdf]

Plasmid pT181 Replication is Regulated by Two Countertranscripts
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397100/pdf/pnas00343-0012.pdf]

The mutH Gene Regulates the Replication and Methylation
of the pMB1 Origin
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207916/pdf/jbacter00100-0187.pdf]

Attempt to Construct a Rop+ pUC19 from pBR322 Mutant Lacking
the Tetracycline Resistance Gene
[http://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/10/10-18.pdf]

Origin of Replication

2013-06-07T14:48:20Z

Bgrieshaber:

'''RNA I and RNA II gene and promoter sites in pSB1a2-BR'''

[[File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎]]

This is an link to the document containing the sequences for RNA I and RNA II sites as well as their promoters. The document contains annotations as to the location of each element. *Note that the full length of RNA II is not fully known but is a close approximation.

----
'''Source Literature'''

Replication and Control of Circular Bacterial Plasmids
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC98921/pdf/mr000434.pdf]

Plasmid ColE1 Incompatibility Determined by Interaction of RNA I
with Primer Transcript
[http://www.pnas.org/content/78/10/6096.full.pdf]

Formation of an RNA Primer for Initiation of Replication of ColE1
DNA by ribonuclease H
[http://www.pnas.org/content/77/5/2450.full.pdf]

Control of pMB1 Replication: Inhibition of Primer Formation
by Rop Requires RNA1
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC557524/pdf/emboj00310-0150.pdf

High Copy Number of the pUC Plasmid Results from a Rom/Rop--Suppressible Point Mutation in RNA II.
[http://content.ebscohost.com/pdf9/pdf/1992/ESL/15Nov92/15903115.pdf?T=P&P=AN&K=15903115&S=R&D=eih&EbscoContent=dGJyMMTo50SeprU4yOvsOLCmr0uep65Ssa24SLSWxWXS&ContentCustomer=dGJyMK6nr0W2pq5T69fnhrnb5ofx6gAA]

Control of ColE1 Replication: Low Affinity Specific Binding
of Rop (Rom) to RNAI and RNAII
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC454354/pdf/emboj00139-0256.pdf]

Plasmid pT181 Replication is Regulated by Two Countertranscripts
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397100/pdf/pnas00343-0012.pdf]

The mutH Gene Regulates the Replication and Methylation
of the pMB1 Origin
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC207916/pdf/jbacter00100-0187.pdf]

Attempt to Construct a Rop+ pUC19 from pBR322 Mutant Lacking
the Tetracycline Resistance Gene
[http://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/10/10-18.pdf]

Origin of Replication

2013-06-06T15:21:22Z

Bgrieshaber: Created page with "'''RNA I and RNA II gene and promoter sites in pSB1a2-BR''' File:PSB1A2_(pMB1)-_RNA_1_and_RNA_2_sites.docx‎ This is an link to the document containing the sequences fo..."

Biology

2013-06-06T15:17:42Z

Bgrieshaber:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Sequences]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

'''Topics to Investigate'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

File:PSB1A2 (pMB1)- RNA 1 and RNA 2 sites.docx

2013-06-06T15:02:21Z

Bgrieshaber: