Difference between revisions of "Team:Freiburg/Protocols/Gibson Assembly"

 
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<h1 class="sectionedit1"><a name="gibson_assembly" id="gibson_assembly">Gibson Assembly</a></h1>
 
<h1 class="sectionedit1"><a name="gibson_assembly" id="gibson_assembly">Gibson Assembly</a></h1>

Latest revision as of 07:07, 20 November 2015

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Gibson Assembly

Gibson Assembly is rather novel method for assembling DNA fragments with overlapping overhangs. The operating mode of this method is devided into three major parts:

  1. An exonuclease removes bases from the 5' end of each DNA strand.
  2. Complementary regions of different DNA strands can anneal and a polymerase fills up the gaps.
  3. The fragments are ligated together.

To enable these three steps, DNA strands with compatible ends of about 32 bp are needed. Those can either be incorporated by primer overhangs or by gene synthesis.
A 5 µl mix of the DNA parts that are supposed to be assembled is prepared. Its composition is calculated based on the length and concentration of every single fragment. The insert(s) should at least be contained in a 4 - 8 fold molar amount of the antibiotic resistance containing backbone.

Assembly of DNA fragments with overlapping regions

(adapted from AG Weber protocol)

Material: Gibson Master Mix Aliquots
Time: 90 min


  1. Prepare 5 µl of DNA-Mix (calculate voulumes using the equations below or use the prepared worksheet.
  2. Add the DNA Mix to Gibson Master Mix.
  3. Incubate for 5 min at RT.
  4. Use 5 - 7 µl for transformation of competent E.coli cells.


Calculation of the DNA mix

  • Backbone:
    V (Bb) = 12 ng/kb * l (Bb) / c (Bb)
  • Insert(s):
    V (Ins) = 12 ng/kb * l (Ins) * X / c (Ins)

    • X = Ratio Insert to Backbone
    for example: X = 4, if Ins : Bb = 4 : 1

    A) Gibson Assembly Master Mix

    Work on ice!

    Volume
    Ingredient
    690 µl
    dH2O
    320 µl
    ISO buffer
    160 µl
    Taq ligase (NEB, 40 U/µl)
    20 µl
    Q5 Polymerase (NEB, 2 U/µl)
    10 µl
    T5 Exonuclease (NEB, 0.64 U/µl)*

    * dilute 3.2 µl T5 Exonuclease (10 U/µl) in 46.8 µl 1x T5-buffer

    aliquot á 15 µl

    B) ISO buffer

    Amount
    Ingredient
    		Remarks
    1.5 g
    PEG-8000
    3 ml
    Tris-HCl (1 M, pH 7.5)
    		dissolve 12.1 g Tris in 100 ml dH2O, adjust pH to 7.5 with conc. HCl
    300 µl
    DTT (1 M)
    		dissolve 1.54 g DTT in 10 ml dH2O
    150 µl
    MgCl2 (2 M)
    		dissolve 4.06 g MgCl2 in 10 ml dH2O
    300 µl
    NADNa (100 mM)
    		dissolve 0.02 g NADNa in 300 µl dH2O
    4 x 60 µl
    dNTPs (100 mM, each)
    up to 6 ml
    dH2O

    aliquot á 350 µl