Team:Paris Saclay/Protocols/BioBrick Assembly

From 2014.igem.org

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(Digest reaction)
(Enzymes)
 
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{{Team:Paris_Saclay/protocols_header}}
=BioBrick Assembly=
=BioBrick Assembly=
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===Procedure===
===Procedure===
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====Enzymes====
====Enzymes====
''<u>Note:</u> Manipulate the enzymes with a proper subzero temperature support.''
''<u>Note:</u> Manipulate the enzymes with a proper subzero temperature support.''
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'''Part B placed after Part A:'''
Part A:
Part A:
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* Enzyme B: SpeI
* Enzyme B: SpeI
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Part B with '''Format I''' (Part B placed after Part A):
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Part B with '''Format I'''
* Enzyme A: EcoRI
* Enzyme A: EcoRI
* Enzyme B: XbaI
* Enzyme B: XbaI
-
Part B with '''Format II''' (Part B placed before Part A):
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'''Part B placed before Part A:'''
-
* Enzyme A: SpeI
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-
* Enzyme B: PstI
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Part A:
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*Enzyme A: EcoR1
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*Enzyme B: XbaI
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 +
Part B with '''Format II'''
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* Enzyme A: EcoRI
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* Enzyme B: SpeI
====Digest reaction====
====Digest reaction====
#Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
#Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
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## xμl of H<sub>2</sub>0 milliQ (complement)
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## x μl of H<sub>2</sub>0 milliQ (complement)
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## 5μl of buffer (Fast Digest Buffer 10x)
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## y μl of buffer (''Fast Digest Buffer 10x'') (1x final concentration)
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## xμl of '''Part A''' DNA
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## z μl of '''Part A''' DNA
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## 1μl of Enzyme A
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## 1 μl of Enzyme A
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## 1μl of Enzyme B
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## 1 μl of Enzyme B
# Repeat '''step 1''' with '''Part B'''
# Repeat '''step 1''' with '''Part B'''
# Mix gently both tubes
# Mix gently both tubes
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====Segregate process====
====Segregate process====
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# part A
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# If the buffer used has no Loading Dye, put it with 1x final concentration.
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# big box, gel 0.8% agarose (and BET concentration???????)
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# Follow the [http://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Electrophoresis Protocol] with the following parameters:
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# 1 hour
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## Make a gel 0.8% agarose
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# minimal exposure of UV light
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## Use 2% of BET concentration
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# mass of the ependorf (before / after)
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## Use a box with a long height and a double case for the DNA.
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## Run the gel at 100V for one hour
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# Determine the weight of the (Eppendorf) tube prior to use.
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# Minimize UV exposure time to avoid damaging the DNA.
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# Put the DNA fragment with a minimal agarose gel into the tube.
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''<u>Note:</u> Minimize UV exposure time to avoid damaging the DNA.''
====DNA extration from agarose gels====
====DNA extration from agarose gels====
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# Excise DNA fragment / solubilize gel slice
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## Determine the weight of the gel slice by the difference of the tube's weight.
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## For each 100 mg of agarose gel < 2% add 200 μl Buffer NTI.
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## Incubate sample for 5-10 min at 50°C.
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## Vortex the sample briefly every 2-3 min until the gel slice is completely dissolved.
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# Bind DNA
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## Place a NucleSpin Gel and PCR Clean-up Column into a Collection Tube and load up to 700 μl sample.
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## Centrifuge for 30 seconds at 11,000 x g. Discard flow-through.
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## Load remaining sample if necessary and repeat the centrifugation step.
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# Wash silica membrane
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## Add 700 μl Buffer NT3 to the column. Centrifuge for 30 s at 11,000 x g. Discard flow-through.
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# Dry silica membrane
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## Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
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# Elute DNA
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## Place the column into a new 1.5 ml micro centrifuge tube. Add 15-30 μl Buffer NE and incubate at room temperature (18-25 °C) for 1 min.
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## Centrifuge for 1 min at 11,000 x g.
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====Ligation====
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{{Team:Paris_Saclay/protocols_footer}}
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====Ligation====
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Latest revision as of 21:57, 17 October 2014

Contents

BioBrick Assembly

This protocol is based on the original 3A Assembly from iGEM. We modified it to a assembly that places the core of one BioBrick - here called Part A - into another BioBrick - here called Part B.

TODO: Process illustration [Format I and Format II]

Procedure

Enzymes

Note: Manipulate the enzymes with a proper subzero temperature support.

Part B placed after Part A:

Part A:

  • Enzyme A: EcoRI
  • Enzyme B: SpeI

Part B with Format I

  • Enzyme A: EcoRI
  • Enzyme B: XbaI

Part B placed before Part A:

Part A:

  • Enzyme A: EcoR1
  • Enzyme B: XbaI

Part B with Format II

  • Enzyme A: EcoRI
  • Enzyme B: SpeI

Digest reaction

  1. Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
    1. x μl of H20 milliQ (complement)
    2. y μl of buffer (Fast Digest Buffer 10x) (1x final concentration)
    3. z μl of Part A DNA
    4. 1 μl of Enzyme A
    5. 1 μl of Enzyme B
  2. Repeat step 1 with Part B
  3. Mix gently both tubes
  4. Incubate at 37°C for one hour
  5. Store Part B at -20°C.

Segregate process

  1. If the buffer used has no Loading Dye, put it with 1x final concentration.
  2. Follow the Electrophoresis Protocol with the following parameters:
    1. Make a gel 0.8% agarose
    2. Use 2% of BET concentration
    3. Use a box with a long height and a double case for the DNA.
    4. Run the gel at 100V for one hour
  3. Determine the weight of the (Eppendorf) tube prior to use.
  4. Minimize UV exposure time to avoid damaging the DNA.
  5. Put the DNA fragment with a minimal agarose gel into the tube.

Note: Minimize UV exposure time to avoid damaging the DNA.

DNA extration from agarose gels

  1. Excise DNA fragment / solubilize gel slice
    1. Determine the weight of the gel slice by the difference of the tube's weight.
    2. For each 100 mg of agarose gel < 2% add 200 μl Buffer NTI.
    3. Incubate sample for 5-10 min at 50°C.
    4. Vortex the sample briefly every 2-3 min until the gel slice is completely dissolved.
  2. Bind DNA
    1. Place a NucleSpin Gel and PCR Clean-up Column into a Collection Tube and load up to 700 μl sample.
    2. Centrifuge for 30 seconds at 11,000 x g. Discard flow-through.
    3. Load remaining sample if necessary and repeat the centrifugation step.
  3. Wash silica membrane
    1. Add 700 μl Buffer NT3 to the column. Centrifuge for 30 s at 11,000 x g. Discard flow-through.
  4. Dry silica membrane
    1. Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
  5. Elute DNA
    1. Place the column into a new 1.5 ml micro centrifuge tube. Add 15-30 μl Buffer NE and incubate at room temperature (18-25 °C) for 1 min.
    2. Centrifuge for 1 min at 11,000 x g.

Ligation