Team:St Pauls London/Protocols

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SAFETY
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===Protocols===
===Protocols===
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All protocols were carried out following safety proceedures as detailed on the [[Team:St_Pauls_London/Safety|safety page]]. The methods used were commercially approved as mentioned below.
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'''QIAGEN approved Plasmid DNA Extraction (a.k.a. The Miniprep):'''
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All buffers used in this method were Biorad-supplied.
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1) Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature (15-25°C).
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2) Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
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3) Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
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4) Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
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5) Centrifuge for 10 min at 13,000 rpm in a table top microcentrifuge.
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6) Apply the supernatant from Step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.
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7) Was the QIAprep spin column by adding 500 μl Buffer PB. Centrifuge for 30-60s and discard the flow through.
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8) Wash the QIAprep spin column to the collection tube.
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9) Centrifuge for 1 min to remove residual wash buffer.
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10) Place the QIAprep column in a clean 1.5 microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (or distilled water) to the centre of the QIAprep spin column, let it stand for 1 min, then centrifuge for 1 min.
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'''NEB approved Transformation (High Efficiency) for 10-beta Competent E. Coli:'''
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1) Thaw a tube of NEB 10-beta Competent E. Coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 μl of cells into a transformation tube on ice.
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2) Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. '''Do not vortex. '''
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3) Place the mixture on ice for 30 mins. Do not mix.
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4) Heat shock at exactly 42°C for exactly 30s. Do not mix.
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5) Place on ice for 5 mins. Do not mix.
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6) Pipette 950 μl of room temperature SOC into the mixture.
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7) Place at 37°C for 60 mins. Shake vigorously or rotate.
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8) Warm selection plates to 37°C.
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9) Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
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10) Spread 50-100 μl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
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'''Restriction Digest and Ligation Techniques:'''
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Originals can be found [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf here].
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Summary:
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1) Start with two BioBrick parts and a BioBrick destinations plasmid. The destinations plasmid contains a toxic gene in the Biobrick cloning site and a different antibiotic resistance marker to the upstream and downstream parts.
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2) Digest each of the parts with the appropriate restriction enzymes.
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3) Mix the digests together and perform a ligation step. One of the ligation products formed will be the correctly assembled composite part in the destinations plasmid. You can use the ligation mix to transform competent cells with the new composite part.

Latest revision as of 12:11, 7 June 2013

Protocols

All protocols were carried out following safety proceedures as detailed on the safety page. The methods used were commercially approved as mentioned below.


QIAGEN approved Plasmid DNA Extraction (a.k.a. The Miniprep):

All buffers used in this method were Biorad-supplied.

1) Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature (15-25°C).

2) Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.

3) Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.

4) Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

5) Centrifuge for 10 min at 13,000 rpm in a table top microcentrifuge.

6) Apply the supernatant from Step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.

7) Was the QIAprep spin column by adding 500 μl Buffer PB. Centrifuge for 30-60s and discard the flow through.

8) Wash the QIAprep spin column to the collection tube.

9) Centrifuge for 1 min to remove residual wash buffer.

10) Place the QIAprep column in a clean 1.5 microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (or distilled water) to the centre of the QIAprep spin column, let it stand for 1 min, then centrifuge for 1 min.


NEB approved Transformation (High Efficiency) for 10-beta Competent E. Coli:

1) Thaw a tube of NEB 10-beta Competent E. Coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 μl of cells into a transformation tube on ice.

2) Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

3) Place the mixture on ice for 30 mins. Do not mix.

4) Heat shock at exactly 42°C for exactly 30s. Do not mix.

5) Place on ice for 5 mins. Do not mix.

6) Pipette 950 μl of room temperature SOC into the mixture.

7) Place at 37°C for 60 mins. Shake vigorously or rotate.

8) Warm selection plates to 37°C.

9) Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

10) Spread 50-100 μl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.



Restriction Digest and Ligation Techniques:

Originals can be found [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf here].

Summary:

1) Start with two BioBrick parts and a BioBrick destinations plasmid. The destinations plasmid contains a toxic gene in the Biobrick cloning site and a different antibiotic resistance marker to the upstream and downstream parts.

2) Digest each of the parts with the appropriate restriction enzymes.

3) Mix the digests together and perform a ligation step. One of the ligation products formed will be the correctly assembled composite part in the destinations plasmid. You can use the ligation mix to transform competent cells with the new composite part.

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