Team:Paris Saclay/Protocols/BioBrick Assembly

From 2014.igem.org

(Difference between revisions)
(Digest reaction)
(Procedure)
Line 36: Line 36:
====Segregate process====
====Segregate process====
# part A
# part A
-
# big box, gel 0.8% agarose (and BET concentration???????)
+
# Follow the [http://2014.igem.org/Team:Paris_Saclay/Protocols/Gel_Electrophoresis Electrophoresis Protocol] with the following parameters:
-
# 1 hour
+
## Make a gel '''0.8%''' agarose
 +
## Use xxx of BET concentration
 +
## Use a box with a such long height and a double case for the DNA.
 +
## Run the gel at 100V for one hour
# minimal exposure of UV light
# minimal exposure of UV light
# mass of the ependorf (before / after)
# mass of the ependorf (before / after)

Revision as of 16:15, 4 August 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 A:

  • Enzyme A: EcoRI
  • Enzyme B: SpeI

Part B with Format I (Part B placed after Part A):

  • Enzyme A: EcoRI
  • Enzyme B: XbaI

Part B with Format II (Part B placed before Part A):

  • Enzyme A: SpeI
  • Enzyme B: PstI

Digest reaction

  1. Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
    1. xμl of H20 milliQ (complement)
    2. 5μl of buffer (Fast Digest Buffer 10x)
    3. xμl of Part A DNA
    4. 1μl of Enzyme A
    5. 1μl 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. part A
  2. Follow the Electrophoresis Protocol with the following parameters:
    1. Make a gel 0.8% agarose
    2. Use xxx of BET concentration
    3. Use a box with a such long height and a double case for the DNA.
    4. Run the gel at 100V for one hour
  3. minimal exposure of UV light
  4. mass of the ependorf (before / after)

DNA extration from agarose gels

Ligation