Prather:Gibson CBA

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==Gibson Chew Back And Anneal Assembly:  One Step Isothermal==
==Gibson Chew Back And Anneal Assembly:  One Step Isothermal==
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Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes. In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment).  These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase.  In theory, many fragments can be assembled simultaneously (5, 4 inserts + backbone, have been documented in the [[Prather:Gibson CBA#References|initial report]]) into a single product of up to ~100 kb+.  The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.
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==Reagents==
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==Method==
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===Reagents===
   
   
'''5x Isothermal Reaction Mix''' <br />
'''5x Isothermal Reaction Mix''' <br />
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:3 ml 1 M Tris-Hcl (pH 7.5)<br />
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:3 ml   1 M Tris-Hcl (pH 7.5)<br />
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:300 ul 1 M MgCl2<br />
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:300 μL 1 M MgCl2<br />
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:60 ul 100 mM dGTP<br />
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:60 μL  100 mM dGTP<br />
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:60 ul 100 mM dATP<br />
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:60 μL 100 mM dATP<br />
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:60 ul 100 mM dTTP<br />
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:60 μL 100 mM dTTP<br />
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:60 ul 100 mM dCTP<br />
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:60 μL 100 mM dCTP<br />
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:300 ul 1 M DTT<br />
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:300 μL 1 M DTT<br />
:1.5 g PEG-8000<br />
:1.5 g PEG-8000<br />
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:300 ul 100 mM NAD<br />
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:300 μL 100 mM NAD<br />
:<ins>balance  ddH2O</ins> <br />
:<ins>balance  ddH2O</ins> <br />
:6 ml Total
:6 ml Total
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'''Assembly Master Mix'''<br />
'''Assembly Master Mix'''<br />
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:320 ul 5X Isothermal Master Mix
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:320 μL  5X Isothermal Master Mix
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:0.64 ul 10 U/ul T5 exonuclease
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:0.64 μL 10 U/μL  T5 exonuclease
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:20 ul         2 U/ul Phusion DNA Pol
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:20 μL  2 U/μL  Phusion DNA Pol
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:0.16 ul 40 000 U/ul Taq DNA Ligase
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:0.16 μL 40 000 U/μL Taq DNA Ligase
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:<ins>860 ul ddH2O</ins>
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:<ins>860 μL ddH2O</ins>
:1.2 ml Total
:1.2 ml Total
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Aliquoted reaction and master mixes are stable at -20C and can withstand several freeze thaw cycles.
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Aliquoted reaction and master mixes are stable at -20°C and can withstand several freeze thaw cycles.
===Protocol===
===Protocol===
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#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
#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
#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
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#Incubate at 50 C for 15-60 min (60 min optimal).
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#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.
<br />
<br />
 +
==Comments==
==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)
# 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)
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# Have successfully used for a two way and three way ligation
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# The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies.  I've gone as high as 170 ng without any ill effects. (KS)
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#There is a potential for point mutations at the DNA boundaries which has yet to be quantified.  Paper suggests 1 every 50 assemblies or so so sequence to verify interfaces or leave spacers at the interfaces to 'absorb' these errors
+
# Have successfully used for a two way and three way ligation (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 contamination) but also has more false positives (PCR template plasmid).  False positive could be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
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# There is a potential for mutations at the DNA boundaries which has yet to be quantified.  Paper suggests 1 every 50 assemblies or so.  Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (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)
+
# I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies.  PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid).  False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
 +
#  I once 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 less overlap if desired (KS)
<br />
<br />
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==References==
==References==
<biblio>
<biblio>
#Gibson-nmeth-2009 pmid=19363495
#Gibson-nmeth-2009 pmid=19363495
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//Isothermal assembly method
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#Gibson-science-2008 pmid=18218864
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//Two step thermocycled assembly variant
</biblio>
</biblio>
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<br />
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----
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Current revision

Contents

Gibson Chew Back And Anneal Assembly: One Step Isothermal

Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes. In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment). These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase. In theory, many fragments can be assembled simultaneously (5, 4 inserts + backbone, have been documented in the initial report) into a single product of up to ~100 kb+. The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.

Method

Reagents

5x Isothermal Reaction Mix

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


Assembly Master Mix

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


Aliquoted reaction and master mixes are stable at -20°C 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. The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects. (KS)
  3. Have successfully used for a two way and three way ligation (KS)
  4. There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
  5. I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
  6. I once 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 less overlap if desired (KS)


References

  1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495. PubMed HubMed [Gibson-nmeth-2009]
    Isothermal assembly method

  2. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:18218864. PubMed HubMed [Gibson-science-2008]
    Two step thermocycled assembly variant

All Medline abstracts: PubMed HubMed



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