Prather:Gibson CBA

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Line 21: Line 21:
:320 ul 5X Isothermal Master Mix
:320 ul 5X Isothermal Master Mix
:0.64 ul 10 U/ul T5 exonuclease
:0.64 ul 10 U/ul T5 exonuclease
-
:20 ul 2 U/ul Phusion DNA Pol
+
:20 ul         2 U/ul Phusion DNA Pol
:0.16 ul 40 000 U/ul Taq DNA Ligase
:0.16 ul 40 000 U/ul Taq DNA Ligase
:<ins>860 ul ddH2O</ins>
:<ins>860 ul ddH2O</ins>
Line 29: Line 29:
Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.
Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.
-
'''Protocol'''
+
===Protocol===
#PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
#PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
#Thaw assembly master mix and keep on ice until ready to be used
#Thaw assembly master mix and keep on ice until ready to be used
Line 35: Line 35:
#Incubate at 50 C for 15-60 min (60 min optimal).
#Incubate at 50 C for 15-60 min (60 min optimal).
#Transform cells with no more than 1 ul of assembly mixture.
#Transform cells with no more than 1 ul of assembly mixture.
 +
 +
===Comments===
 +
----
 +
# When preparing the isothermal reaction mix, add the PEG slowly to liquid.  If added too quickly it will form a plug which will make mixing difficult (KS)
 +
# I have used PCRs as is (with PCR cleanup only) and with gel extracted DNA.  PCR Cleanup gives more colonies (more DNA, better quality (no agarose, QG contam) but also has more false positives (PCR template plasmid).  False positive could be alleviated by DpnI treatment inf gel extraction is not used but I haven't tested this yet (KS).
 +
#  I inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids.  Possible to use shorter primers if desired (KS)
 +
 +
===References===
 +
Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009. [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html ||doi:10.1038/nmeth.1318]

Revision as of 18:31, 8 August 2010

Contents

Gibson Chew Back And Anneal Assembly: One Step Isothermal

Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009.

Reagents

5x Isothermal Reaction Mix

3 ml 1 M Tris-Hcl (pH 7.5)
300 ul 1 M MgCl2
60 ul 100 mM dGTP
60 ul 100 mM dATP
60 ul 100 mM dTTP
60 ul 100 mM dCTP
300 ul 1 M DTT
1.5 g PEG-8000
300 ul 100 mM NAD
balance ddH2O
6 ml Total


Assembly Master Mix

320 ul 5X Isothermal Master Mix
0.64 ul 10 U/ul T5 exonuclease
20 ul 2 U/ul Phusion DNA Pol
0.16 ul 40 000 U/ul Taq DNA Ligase
860 ul ddH2O
1.2 ml Total


Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.

Protocol

  1. PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
  2. Thaw assembly master mix and keep on ice until ready to be used
  3. Mix 15 ul of assembly mixture with 5 ul total of cleaned PCR product (PCR Cleanup Kit or Gel Extraction) keeping DNA inserts in equimolar amounts
  4. Incubate at 50 C for 15-60 min (60 min optimal).
  5. Transform cells with no more than 1 ul of assembly mixture.

Comments


  1. When preparing the isothermal reaction mix, add the PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult (KS)
  2. I have used PCRs as is (with PCR cleanup only) and with gel extracted DNA. PCR Cleanup gives more colonies (more DNA, better quality (no agarose, QG contam) but also has more false positives (PCR template plasmid). False positive could be alleviated by DpnI treatment inf gel extraction is not used but I haven't tested this yet (KS).
  3. I inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use shorter primers if desired (KS)

References

Gibson, DG et al. Nature Methods, 6 (5) 343-345, 2009. ||doi:10.1038/nmeth.1318

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