Team:Brown-Stanford/Lab/Protocols/Gibson

From 2011.igem.org

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{{:Team:Brown-Stanford/Templates/Protocol}}
{{:Team:Brown-Stanford/Templates/Protocol}}
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<html><h1>Protocol Code: AY1</h1></html>
<html><h1>Protocol Code: AY1</h1></html>
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''5x isothermal buffer''
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'''5x isothermal buffer'''
*0.75g - 25% PEG-8000
*0.75g - 25% PEG-8000
*1500 &#181;L - 500 mM Tris-HCl pH 7.5
*1500 &#181;L - 500 mM Tris-HCl pH 7.5
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Aliquoted reaction and master mixes are stable at -20°C and can withstand several freeze thaw cycles.
Aliquoted reaction and master mixes are stable at -20°C and can withstand several freeze thaw cycles.
==='''Protocol'''===
==='''Protocol'''===
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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
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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.
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#Transform cells with no more than 1 ul of assembly mixture.
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Comments
+
==='''Comments'''===
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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)
-
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)
+
#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)
-
Have successfully used for a two way and three way ligation (KS)
+
#Have successfully used for a two way and three way ligation (KS)
-
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)
+
#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 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 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)
+
#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)
{{:Team:Brown-Stanford/Templates/Foot}}
{{:Team:Brown-Stanford/Templates/Foot}}

Latest revision as of 18:17, 28 September 2011

Brown-Stanford
iGEM

Back to
iGEM HQ

Protocol Code: AY1

Gibson Assembly

5x isothermal buffer

  • 0.75g - 25% PEG-8000
  • 1500 µL - 500 mM Tris-HCl pH 7.5
  • 75 µL - 50mM MgCl2
  • 150 µL - 50mM DTT
  • 30 µL - 1mM dATP
  • 30 µL - 1mM dTTP
  • 30 µL - 1mM dCTP
  • 30 µL - 1mM dGTP
  • 300 µL - 5mM NAD
  • Nuclease-free water to fill to 3000µL

Total - 3000 µL


Gibson Master Mix

  • 50 µL - Taq ligase (40u/µL)
  • 100 µL - 5x isothermal buffer
  • 2 µL - T5 exonuclease (1u/µL)
  • 6.25 µL - Phusion polymerase (2u/µL)
  • 216.75 µL - Nuclease-free water

Total - 375 µL

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)

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