GcatWiki - User contributions [en]

DNA Sequencing at Eurofins

2018-06-21T15:00:05Z

Eckdahl:

Prepare template and primer solutions at the concentrations listed at the bottom of this page.

# Grab the appropriate number of bar-code tubes to be sent off for sequencing.
# Record the code on each tube and what clone will be in it inside your lab notebook.
# Add your sample to the correct bar-code tube.

'''How to Prepare'''

1.Prepare aqueous template and primer solution at the concentrations listed below.

2. Add 8ul template + 4ul (2uM) to each tube by inserting your pipet tip through the split seal (total volume = 12ul)

3. Place tubes into a Blue Bag or other ziplock bag

4. Ship using a UPS padded envelope from the Overnight Kit or place Blue Bag in a near-by Eurofins Genomics Dropbox

PCR < 300 Bp (10-20ng/ul)

PCR 300-1000 Bp (20-40ng/ul)

PCR >1000 Bp (40-60ng/ul)

Plasmids all lenghts (100-200ng/ul)

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-18T18:25:04Z

Eckdahl:

Clean and Concentrate Protocol

Ratios (DNA Binding Buffer : Sample)

2:1 plasmid, genomic DNA

5:1 PCR product, DNA fragment

7:1 ssDNA

When using plasmid DNA that has been through Miniprep Protocol.

1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200µl DNA Binding Buffer : 100µl plasmid DNA)

2. Mix gently by vortex.

3. Transfer mixture to Zymo-SpinTM Column in a Collection Tube.

4. Centrifuge for 30 seconds (11,000 - 16,000 rpm) and discard the flow through.

5. Add 200µl of DNA Wash Buffer to the column. Centrifuge for 30 seconds.

6. REPEAT Step 5.

7. Add 25µl of dH20 (55̊C) to the column. Let sit at room temperature for approximately 2 minutes.

8. Transfer column to 1.5ml microcentrifuge tube. Centrifuge for 30 seconds.

Results:

Start: 65.5ng/µl

After Clean and Concentrate: 50ng/µl

Yield: 76.3%

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-18T18:23:55Z

Eckdahl:

Clean and Concentrate Protocol

Ratios (DNA Binding Buffer : Sample)

2:1 plasmid, genomic DNA

5:1 PCR product, DNA fragment

7:1 ssDNA

When using plasmid DNA that has been through Miniprep Protocol.

1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200µl DNA Binding Buffer : 100µl plasmid DNA)

2. Mix gently by vortex.

3. Transfer mixture to Zymo-SpinTM Column in a Collection Tube.

4. Centrifuge for 30 seconds and discard the flow through.

5. Add 200µl of DNA Wash Buffer to the column. Centrifuge for 30 seconds.

6. REPEAT Step 5.

7. Add 25µl of dH20 (55̊C) to the column. Let sit at room temperature for approximately 2 minutes.

8. Transfer column to 1.5ml microcentrifuge tube. Centrifuge for 30 seconds.

Results:

Start: 65.5ng/µl

After Clean and Concentrate: 50ng/µl

Yield: 76.3%

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-18T18:23:36Z

Eckdahl:

Clean and Concentrate Protocol

Ratios (DNA Binding Buffer : Sample)

2:1 plasmid, genomic DNA
5:1 PCR product, DNA fragment
7:1 ssDNA

When using plasmid DNA that has been through Miniprep Protocol.

1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200µl DNA Binding Buffer : 100µl plasmid DNA)

2. Mix gently by vortex.

3. Transfer mixture to Zymo-SpinTM Column in a Collection Tube.

4. Centrifuge for 30 seconds and discard the flow through.

5. Add 200µl of DNA Wash Buffer to the column. Centrifuge for 30 seconds.

6. REPEAT Step 5.

7. Add 25µl of dH20 (55̊C) to the column. Let sit at room temperature for approximately 2 minutes.

8. Transfer column to 1.5ml microcentrifuge tube. Centrifuge for 30 seconds.

Results:

Start: 65.5ng/µl

After Clean and Concentrate: 50ng/µl

Yield: 76.3%

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-14T20:10:05Z

Eckdahl:

Clean and Concentrate Protocol

When using plasmid DNA that has been through Miniprep Protocol.

1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200µl DNA Binding Buffer : 100µl plasmid DNA)

2. Mix gently by vortex.

3. Transfer mixture to Zymo-SpinTM Column in a Collection Tube.

4. Centrifuge for 30 seconds and discard the flow through.

5. Add 200µl of DNA Wash Buffer to the column. Centrifuge for 30 seconds.

6. REPEAT Step 5.

7. Add 25µl of dH20 (55̊C) to the column. Let sit at room temperature for approximately 2 minutes.

8. Transfer column to 1.5ml microcentrifuge tube. Centrifuge for 30 seconds.

Results:

Start: 65.5ng/µl

After Clean and Concentrate: 50ng/µl

Yield: 76.3%

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-14T20:09:30Z

Eckdahl:

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-14T20:09:18Z

Eckdahl:

Clean and Concentrate Protocol
When using plasmid DNA that has been through Miniprep Protocol.

1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200µl DNA Binding Buffer : 100µl plasmid DNA)

2. Mix gently by vortex.

3. Transfer mixture to Zymo-SpinTM Column in a Collection Tube.

4. Centrifuge for 30 seconds and discard the flow through.

5. Add 200µl of DNA Wash Buffer to the column. Centrifuge for 30 seconds.

6. REPEAT Step 5.

7. Add 25µl of dH20 (55̊C) to the column. Let sit at room temperature for approximately 2 minutes.

8. Transfer column to 1.5ml microcentrifuge tube. Centrifuge for 30 seconds.

Results:

Start: 65.5ng/µl

After Clean and Concentrate: 50ng/µl

Zymo Research Clean and Concentrate for Plasmid DNA

2018-06-14T20:08:36Z

Eckdahl: Created page with "Clean and Concentrate Protocol When using plasmid DNA that has been through Miniprep Protocol. 1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200..."

Clean and Concentrate Protocol
When using plasmid DNA that has been through Miniprep Protocol.
1. Combine 2 volumes of DNA Binding Buffer to each volume of plasmid. (Ex. 200µl DNA Binding Buffer : 100µl plasmid DNA)
2. Mix gently by vortex.
3. Transfer mixture to Zymo-SpinTM Column in a Collection Tube.
4. 4. Centrifuge for 30 seconds and discard the flow through.
5. Add 200µl of DNA Wash Buffer to the column. Centrifuge for 30 seconds.
6. REPEAT Step 5.
7. Add 25µl of dH20 (55̊C) to the column. Let sit at room temperature for approximately 2 minutes.
8. Transfer column to 1.5ml microcentrifuge tube. Centrifuge for 30 seconds.

Results:
Start: 65.5ng/µl
After Clean and Concentrate: 50ng/µl

MWSU protocols

2018-06-14T20:08:17Z

Eckdahl:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

[[Gel Purification New England BioLabs Monarch]]

'''PCR'''

[[iPCR]]

[[Gradient/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]]

[[Zymo Research Clean and Concentrate for Plasmid DNA]]

[[Golden Gate Assembly Protocol]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[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]]

[[SOC Protocol for Transformations]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[DNA Sequencing at Eurofins]]

[[What to do with a new clone]]

[[How to make a new Registry page]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

MWSU protocols

2017-02-16T15:27:58Z

Eckdahl:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

[[Gel Purification New England BioLabs Monarch]]

'''PCR'''

[[iPCR]]

[[Gradient/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]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[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]]

[[SOC Protocol for Transformations]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[DNA Sequencing at Eurofins]]

[[What to do with a new clone]]

[[How to make a new Registry page]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

DNA Sequencing at Eurofins

2017-02-16T15:24:22Z

Eckdahl: Created page with "# Prepare template and primer solutions at the concentrations listed at the bottom of this page. # Grab the appropriate number of bar-code tubes to be sent off for sequencing...."

# Prepare template and primer solutions at the concentrations listed at the bottom of this page.
# Grab the appropriate number of bar-code tubes to be sent off for sequencing.
# Record the code on each tube and what clone will be in it inside your lab notebook.
# Get 8 microliters of your sample and add it to the correct bar-code tube. This can be done by putting the tip through the septum and ejecting the sample onto the inner wall of the tube.

MWSU protocols

2017-02-15T21:14:27Z

Eckdahl:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Gradient/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]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[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]]

[[SOC Protocol for Transformations]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[DNA Sequencing at Eurofins]]

[[What to do with a new clone]]

[[How to make a new Registry page]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

How to make a new Registry page

2017-02-15T19:57:58Z

Eckdahl: Created page with "'''Making a Registry Page''' # Go to Registry of Biological Parts then login as the correct username # Hit the tools tab and click add a part # Click add a Basic Part # Allow..."

'''Making a Registry Page'''

# Go to Registry of Biological Parts then login as the correct username
# Hit the tools tab and click add a part
# Click add a Basic Part
# Allow edits for Eckdahl Lab and for Davidson Lab (Just check Eckdahl's box and check one of Davidson's box)
# Copy what is directly under "next available part" (should be some letters followed by a sequence of numbers and if doing the Pupp promoter experiment, your part name is written on the Annealed Oligos Page hanging up in the lab)
# Paste this under Selected Part name
# Select your part type (ex. in this case for the Pupp promoter it is regulatory)
# Enter a short description (this is what people around the world will see) include what you did.
# If you get stumped on what to write go to Davidson's upp promoters and see what they wrote.
# Then go into detail on what you did and how it effected this sequence in the long description
# Enter in the source (ex. in the Pupp promoter experiment, the source was put as: Synthetic Oligonucleotides cloned into pClone Red
# Enter "None" in design consideration
# Click go on to enter sequence... annotations
# Edit your designer name (it should be your full name along with anybody else involved in the experiment)
# Click go to Part tools, then click edit sequence and features.
# Click the little blue edit button
# Enter in the top strand of your sequence leaving out the first four letters and the last four letters because those are just the sticky ends
# Click part tools and select edit sequence and features then click add a feature
# When adding a feature this will want you to select a type (ex. In PuppDel-10 there was a mutated -10 element so mutated was selected)
# Then add the label name
# Count left to right where the sequence starts and where it ends. Place those numbers on the start and end columns. The direction will be fwd.
# There may be more than one feature in the sequence so repeat the same procedure for other features (ex. PuppDel-10 had two features -35 element and the -10 element)
# Then save this and your entry will be added

MWSU protocols

2017-02-15T19:05:40Z

Eckdahl:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Gradient/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]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[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]]

[[SOC Protocol for Transformations]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

[[How to make a new Registry page]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

Pouring an Agarose Gel

2017-02-15T19:03:52Z

Eckdahl:

'''Pouring an Agarose Gel'''

# Add 35 mL of 1X TAE buffer to a 125 ml flask
# Add agarose (0.35 grams agarose for 1% gel).
# Microwave for 50 seconds
# Swirl and microwave for 5 seconds - repeat 3 times
# Place flask in 55 C water bath to cool
# Add 5 µL of 10 mg/ml Ethidium Bromide (CAUTION: WEAR GLOVES)
# Swirl to mix and pour into gel tray with comb

Pouring an Agarose Gel

2017-02-15T19:03:40Z

Eckdahl:

'''Pouring an Agarose Gel''

# Add 35 mL of 1X TAE buffer to a 125 ml flask
# Add agarose (0.35 grams agarose for 1% gel).
# Microwave for 50 seconds
# Swirl and microwave for 5 seconds - repeat 3 times
# Place flask in 55 C water bath to cool
# Add 5 µL of 10 mg/ml Ethidium Bromide (CAUTION: WEAR GLOVES)
# Swirl to mix and pour into gel tray with comb

Pouring an Agarose Gel

2017-02-15T19:02:59Z

Eckdahl:

Pouring an Agarose Gel

2017-02-15T19:01:28Z

Eckdahl:

'''Pouring an Agarose Gel'''

#Go to http://www.bio.davidson.edu/courses/Molbio/Protocols/molwt.html to determine the percentage of agarose and calculate amount of agarose for 35 ml.

# Weigh out agarose in 125 ml flask (eg. 0.35 grams agarose for 1% gel).
# Add 35 mL of 1X TAE buffer
# Microwave for 50 seconds
# Swirl and microwave for 5 seconds - repeat 3 times
# Place flask in 55 C water bath to cool
# Add 5 µL of 10 mg/ml Ethidium Bromide (CAUTION: WEAR GLOVES)
# Swirl to mix and pour into gel tray with comb

Golden Gate Assembly Protocol

2017-02-09T16:15:41Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (20 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6.9 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

:0.5 µL T4 DNA Ligase to the mixture

:10.9 µL total volume

Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:

:• 30 cycles

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 37°C for 15 minutes (after 30 cycles)

:• 10°C holding temperature

Turn on the thermal cycler machine. Put tube into machine. For BsmBI GGA, program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 10°C holding temperature

After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Golden Gate Assembly Protocol

2017-02-09T16:12:00Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (20 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:

:• 30 cycles

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 37°C for 15 minutes (after 30 cycles)

:• 10°C holding temperature

Turn on the thermal cycler machine. Put tube into machine. For BsmBI GGA, program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 10°C holding temperature

After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Golden Gate Assembly Protocol

2017-02-09T15:39:25Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (20 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:

:• 20 cycles

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

Turn on the thermal cycler machine. Put tube into machine. For BsmBI GGA, program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 10°C holding temperature

After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Ligation and Transformation

2017-02-02T21:39:33Z

Eckdahl:

'''Ligation'''

#Plan to do a ligation with the vector only and one with vector + insert.
#Dilute vector to 20 ng/ul for use in the ligation, use Millipore pure dH2O.
#Calculate the amount of insert needed with the following formula:
10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng
# Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube
# Add 1 ul 10X Ligation buffer and mix with pipetting.
# Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase]and mix with pipetting.
# Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL.
#Let stand 5 min at room temperature.
#Thaw competent cells on ice (eg. 100 ul of [http://www.zymoresearch.com/content/z-competent-e-coli-t3003-t3005-t3007-t3009-t3011-t3013-t3015-t3017 Z-competent JM109]).
#Heat ligation at 65 C for 10 minutes.
#Place ligation tubes on ice for 2 minutes.

'''Transformation'''

#Before doing the actual transformation procedure, get your plates that you intend to use and place them in the incubator, agar side down so that they can warm up while getting the bacteria prepped.
#Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates.
#Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation.
#Let stand on ice for 5 minutes.
#Spread your mix onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
#Place your plates into the incubator.

Ligation and Transformation

2017-02-02T21:33:08Z

Eckdahl:

'''Ligation'''

#Plan to do a ligation with the vector only and one with vector + insert.
#Dilute vector to 20 ng/ul for use in the ligation, use Millipore pure dH2O.
#Calculate the amount of insert needed with the following formula:
10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng
# Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube
# Add 1 ul 10X Ligation buffer and mix with pipetting.
# Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase]and mix with pipetting.
# Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL.
#Let stand 5 min at room temperature.
#Thaw competent cells on ice (eg. 100 ul of [http://www.zymoresearch.com/content/z-competent-e-coli-t3003-t3005-t3007-t3009-t3011-t3013-t3015-t3017 Z-competent JM109]).
#Heat ligation at 65 C for 10 minutes.
#Place ligation tubes on ice for 2 minutes.

'''Transformation'''

#Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates.
#Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation.
#Let stand on ice for 5 minutes.
#Spread onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
#Place your plates into the incubator.

Golden Gate Assembly Protocol

2016-03-01T19:44:04Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:

:• 20 cycles

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

Turn on the thermal cycler machine. Put tube into machine. For BsmBI GGA, program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 10°C holding temperature

After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Golden Gate Assembly Protocol

2016-03-01T19:22:26Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:

:• 20 cycles

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

Turn on the thermal cycler machine. Put tube into machine. For BsmBI GGA, program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

After GGA, the DNA is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Golden Gate Assembly Protocol

2016-03-01T19:08:28Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the thermal cycler machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Golden Gate Assembly Protocol

2016-03-01T19:06:53Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the PCR machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Ethanol Precipitation of Vector DNA

2015-06-04T14:53:05Z

Eckdahl:

# After digestion with restriction enzymes (ex. Normally a 40 µL digestion)
# Increase volume to 90 µL with dH20
# Add 10 µL of 3M NaOAc pH 5.2
# Add 2X the volume of ethanol (ex. 200 µL of ethanol)
# Vortex and put in -80°C freezer for 15 minutes
# Centrifuge for 30 minutes (Place the tube hinge out in the microcentrifuge to know where the pellet is)
# Dump off the liquid and allow to dry, either sitting on the bench or with the lyophilizer

Gradient/Standard PCR

2015-06-02T13:39:26Z

Eckdahl: Created page with " Gradient/ Standard PCR 1. Create standard PCR mix. 10ul 2X GoTaq Green PCR mix 1ul forward primer (10mMl) 1ul reverse primer (10mM) 7ul dH2O 19ul Total Add 1ul of ..."

Gradient/ Standard PCR

1. Create standard PCR mix.

10ul 2X GoTaq Green PCR mix

1ul forward primer (10mMl)

1ul reverse primer (10mM)

7ul dH2O

19ul Total

Add 1ul of template DNA

2. Using the table below, program the gradient (Δ°C) to match the desired temperature range from the standard temperature. ± values are approximate when indicated with ~.

Δ°C ±°C from Standard Temperature

Δ12.0°C ±5°C

Δ14°C ~±6°C

Δ17°C ~±7°C

Δ19°C ~±8°C

Δ21°C ~±9°C

Δ24°C ±10°C

Δ27°C ~±11°C

Δ29°C ~±12°C

Δ30°C ±12.5°C

Thermal Profile

• 1 cycle of 94°C for 4min

• 30 cycles of 94°C for 15s, Annealing temperature/Gradient (Δ°C) for 15s, 74°C for 30s

• 1 cycle of 74°C for 4min

(The annealing temperature should be about 5°C below the lower of the Tm values of the 2 primers)

This page was last modified on June 1, 2015, at 16:10.

MWSU protocols

2015-06-02T13:33:52Z

Eckdahl:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Gradient/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]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[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]]

[[SOC Protocol for Transformations]]

'''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]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

Standard PCR

2015-06-02T13:31:22Z

Eckdahl:

Gradient/ Standard PCR
1.Create standard PCR mix.

10ul 2X GoTaq Green PCR mix

1ul forward primer (10mMl)

1ul reverse primer (10mM)

7ul dH2O

19ul Total

Add 1ul of template DNA

2. Using the table below, program the gradient (Δ°C) to match the desired temperature range from the standard temperature. ± values are approximate when indicated with ~.

Δ°C ±°C from Standard Temperature
Δ12°C ±5°C

Δ14°C ~±6°C

Δ17°C ~±7°C

Δ19°C ~±8°C

Δ21°C ~±9°C

Δ24°C ±10°C

Δ27°C ~±11°C

Δ29°C ~±12°C

Δ30°C ±12.5°C

Thermal Profile

• 1 cycle of 94°C for 4min

• 30 cycles of 94°C for 15s, Annealing temperature/Gradient (Δ°C) for 15s, 74°C for 30s

• 1 cycle of 74°C for 4min

(The annealing temperature should be about 5°C below the lower of the Tm values of the 2 primers)

This page was last modified on June 1, 2015, at 16:10.

Standard PCR

2015-06-02T13:17:41Z

Eckdahl:

Gradient/ Standard PCR
1. Create standard PCR mix.
a. 10ul 2X GoTaq Green PCR mix
b. 1ul forward primer (10mMl)
c. 1ul reverse primer (10mM)
d. 7ul dH2O
19ul Total
Add 1ul of template DNA
2. Using the table below, program the gradient (Δ°C) to match the desired temperature range from the standard temperature. ± values are approximate when indicated with ~.

Δ°C ±°C from Standard Temperature
Δ12°C ±5°C
Δ14°C ~±6°C
Δ17°C ~±7°C
Δ19°C ~±8°C
Δ21°C ~±9°C
Δ24°C ±10°C
Δ27°C ~±11°C
Δ29°C ~±12°C
Δ30°C ±12.5°C

Thermal Profile
• 1 cycle of 94°C for 4min
• 30 cycles
o 94°C for 15s
o Annealing temperature/Gradient (Δ°C) for 15s
o 74°C for 30s
• 1 cycle of 74°C for 4min
(The annealing temperature should be about 5°C below the lower of the Tm values of the 2 primers)
This page was last modified on June 1, 2015, at 16:10.

Golden Gate Assembly Single Molecule Protocol

2015-06-01T19:32:53Z

Eckdahl: Created page with "Golden Gate Assembly Single Molecule Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL 10X Promega Ligase Buffer :7 µL dH2O :0.5 µL Restrictio..."

Golden Gate Assembly Single Molecule Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL 10X Promega Ligase Buffer

:7 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL volume

Turn on the PCR machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

MWSU protocols

2015-06-01T19:28:48Z

Eckdahl:

'''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]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[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]]

[[SOC Protocol for Transformations]]

'''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]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

Golden Gate Assembly Protocol

2015-06-01T19:27:45Z

Eckdahl: Created page with "Golden Gate Assembly Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL promoter or other insert :1 µL 10X Promega Ligase Buffer :6 µL dH2O :0..."

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL total volume

Turn on the PCR machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

MWSU protocols

2015-06-01T19:27:32Z

Eckdahl:

'''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]]

[[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]]

[[SOC Protocol for Transformations]]

'''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]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

Golden Gate Assembly Protocol for BsmB1

2015-06-01T19:26:11Z

Eckdahl:

Golden Gate Assembly Protocol for BsmB1

2015-06-01T19:25:31Z

Eckdahl:

Golden Gate Assembly Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL promoter or other insert

:1 µL 10X Promega Ligase Buffer

:6 µL dH2O

:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL volume

Turn on the PCR machine. Put tube into machine. Program it for the following cycles:

:• 20 cycles

:• 55°C for 10 minutes

:• 37°C for 1 minute

:• 16°C for 1 minute

:• 22°C holding temperature

This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

Measuring Fluorescence in Bacteria

2015-05-20T14:22:08Z

Eckdahl:

'''Measuring Fluorescence in Bacteria'''

This is the procedure for measuring the function of RFP and GFP in bacterial cultures.
 

#All Wells should contain 250 microliters of cells in broth. Be sure to include wells containing broth with no cells- for negative control.
#Open Gen 5 software by double clicking on the desktop shortcut.
#Click on Existing protocol
#Select Standard Protocol type
##For RFP, select 585 nm (excitation) and 615 nm (emission) for optimal reading (with 100 gain)
##For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
#Select wells filled
#Place the well inside the machine, and Click OK when ready.
#To change color scale, click edit matrix, blue scale, custom, scale drop down menue, custom scale, select colors, and click OK.
#After the machine has run, you can export the data to Excel using the Excel button on the banner of the window that pops up.
##Under data drop down menue, look at ratio or number of bacteria also.

Measuring Fluorescence in Bacteria

2015-05-20T14:21:56Z

Eckdahl:

'''Measuring Fluorescence in Bacteria'''

This is the procedure for measuring the function of RFP and GFP in bacterial cultures.
 

#All Wells should contain 250 microliters of cells in broth. Be sure to include wells containing broth with no cells- for negative control.
#Open Gen 5 software by double clicking on the desktop shortcut.
#Click on Existing protocol
#Select Standard Protocol type
##For RFP, select 585 nm (excitation) and 615 nm (emission) for optimal reading (with 100 gain)

##For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
#Select wells filled
#Place the well inside the machine, and Click OK when ready.
#To change color scale, click edit matrix, blue scale, custom, scale drop down menue, custom scale, select colors, and click OK.
#After the machine has run, you can export the data to Excel using the Excel button on the banner of the window that pops up.
##Under data drop down menue, look at ratio or number of bacteria also.

Fragment Purification

2015-05-19T14:13:42Z

Eckdahl:

'''Fragment Purification from an Agarose Gel'''

[http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel

#With a plastic ruler cut band out of agarose gel and place in 1.5 microcentrifuge tube.
#Add 700 ul of buffer NTI and incubate at least 5 minutes in 55 C water bath, vortexing every minute until gel slice disappears.
#Incubate a dH20 in 55 C water bath for at later use.
#Transfer 700 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 30 seconds at full speed
#Dump the collection tube, transfer the remaining volume to the column, and spin 30 seconds.
#Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column, then spin 30 seconds.
#Repeat step 6.
#Dump collection tube and add 250 ul wash buffer NT3 to column, then spin 30 seconds.
#Repeat step 8.
#Dump the collection tube and spin the column at full speed for 1 minute to dry colum.
#Transfer to a NEW 1.5 ml tube.
#Add 12 ul heated dH2O directly to filter, let stand 30 seconds, and spin 30 seconds.
#Reapply the eluate onto the column, let stand 1 minute, spin 1 minute.
#Measure concentration on Nanodrop

References 2015

2015-02-06T20:42:16Z

Eckdahl:

tunning bacteria through promoter engineering [[Hal Alper]]

References 2015

2015-02-06T20:40:45Z

Eckdahl:

tunning bacteria through promoter engineering [[Media:Hal alper]]

References 2015

2015-02-03T21:20:14Z

Eckdahl: Blanked the page

References 2015

2015-02-03T21:19:49Z

Eckdahl:

[[Media:Vincent JJ Martin.pdf]]

References 2015

2015-02-03T21:18:06Z

Eckdahl:

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids [[File:Vincent JJ Martin]]

References 2015

2015-02-03T21:17:50Z

Eckdahl:

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids [[File:File: Vincent JJ Martin]]

References 2015

2015-02-03T21:16:10Z

Eckdahl:

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids [[File:Vincent JJ Martin]]

References 2015

2015-02-03T21:15:08Z

Eckdahl:

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids [[Media:File:Vincent JJ Martin]]

References 2015

2015-02-03T21:13:32Z

Eckdahl:

Tuning genetic control through promoter engineering [[File: Hal Alper.pdf]]