Janet B. Matsen:Guide to Gibson Assembly

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== Intro ==
== Intro ==
* What is it?
* What is it?
 +
** Important: the efficiency is low, however, you are selecting for complete/circularized products at the transformation step. Usually you will have no shortage of colonies to screen and sequence.
* How does it differ from other cloning?
* How does it differ from other cloning?
* When should I use it?  
* When should I use it?  
Line 10: Line 11:
** Use Gibson Assembly Mix
** Use Gibson Assembly Mix
** Transform
** Transform
-
*** Electroporation is usually used to provide higher yield.  
+
*** Electroporation is usually used to provide higher yield.
-
==Procedure==
+
== Other Resources==
-
=== Make a plasmid map of your design ===
+
* [http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html Guide] by the creaters of Gibthon (software I haven't tried)
-
* This is key.  You will want it for primer design, checking your primers, assessing sequencing reactions, etc.  I use [http://biologylabs.utah.edu/jorgensen/wayned/ape/ APE], open-source software.  See my [[APE use page]]  
+
* I don't like [http://nebuilder.neb.com/# NEBuilder] (web design tool):
 +
** It gave me overlaps of only 20 bp.  I didn't see an option to request longer primers (to get more homology).  When I changed the minimum to 60 bp it added more length to the priming portion of each primer, not the the homology-conferring portion.  Strange...
 +
** It doesn't use backbone primers that confer homology.  It only primes at the border.  I have always used long primers that make homology to my insert.  I am aware that this means I can't have re-usable backbone amplifications, but I feel it could only be advantageous for assembly.  I am asking Rob Egbert if he thinks this matters much.
 +
 
 +
==Protocol (descriptive version)==
 +
* See below for consolidated version.
 +
=== Prepare plasmid maps ===
 +
* Make a plasmid map of what your completed design should look like
 +
** This is key.  You will want it for primer design, checking your primers, assessing sequencing reactions, etc.  I use [http://biologylabs.utah.edu/jorgensen/wayned/ape/ APE], open-source software.  See my [[APE use page]]  
 +
** Make sure each gene has a promoter, RBS, and stop codon. 
 +
** If you aren't familiar with your sequences, make sure the sequence has no stop codons with the frame of reference it starts with.  When downloading a sequence from somewhere like Genbank or getting a plasmid from somebody, this check should be done.
 +
* Make a plasmid map for each segment you will PCR amplify from a template
 +
** Include only the regions that will be present in your final assembly.
 +
** Do not include any homology regions initially.  As you design your primers, make a new copy of each file that shows what the fragment will look like after priming as a PCR product.
=== Design primers ===
=== Design primers ===
* The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.  
* The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.  
Line 31: Line 45:
*I run each PCR at a few annealing temps and DMSO concentrations.  See my sample spreadsheet.  
*I run each PCR at a few annealing temps and DMSO concentrations.  See my sample spreadsheet.  
*[http://www.neb.com/nebecomm/products/productr0176.asp Dpn1] can be added after the PCR is complete to degrade the template DNA.  This will reduce the number of background colonies when you transform.
*[http://www.neb.com/nebecomm/products/productr0176.asp Dpn1] can be added after the PCR is complete to degrade the template DNA.  This will reduce the number of background colonies when you transform.
 +
** you will only get background if the antibiotic marker of the template is that of your design goal.
 +
** gel purification without doing DPN1 digestion usually is sufficient to greatly reduce background.
 +
** here is a sample result of background for a scenario where I used ~0.5 ng of template plasmid per 25 uL of PCR reaction to produce my backbone, then column purified (not gel purified!), and didn't do a DPN1 digestion.  The pink colonies are the plasmid template carrying through the column purification, into the assembly reaction and transformation step.  [[image:transformation reaction 2013_03_12 pg 250 plate 21.jpg|thumb|center|pink colonies in this transformation of a Gibson assembly are carry-through of template from the PCR amplification of the backbone]]
* Run a few uL of each PCR product on a gel to identify rxn conditions that yield a lot of product.
* Run a few uL of each PCR product on a gel to identify rxn conditions that yield a lot of product.
Line 37: Line 54:
**Elute in 30 uL to provide a concentrated product.  
**Elute in 30 uL to provide a concentrated product.  
* You can do a PCR-cleanup instead to get higher yield *if* you run a few uL of the PCR product and it looks totally perfect.  If you have ~ 100 uL of PCR product and the band is strong, I recommend gel purifying anyway.  You will lose some, but the purity of your PCR product will be advantageous for assembly.  
* You can do a PCR-cleanup instead to get higher yield *if* you run a few uL of the PCR product and it looks totally perfect.  If you have ~ 100 uL of PCR product and the band is strong, I recommend gel purifying anyway.  You will lose some, but the purity of your PCR product will be advantageous for assembly.  
 +
** Using Dpn1 on the PCR product is good if you are going to do this, especially for anything that had template with the antibiotic resistance of your design goal, and if it is a backbone amplification.
** Elute in 30 uL.  
** Elute in 30 uL.  
* You will want ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are tolerable.  If the band in your gel is strong and you have ~100 uL of PCR product, yields of ~ 50 ng/uL are more common.
* You will want ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are tolerable.  If the band in your gel is strong and you have ~100 uL of PCR product, yields of ~ 50 ng/uL are more common.
=== Gibson assembly reaction ===
=== Gibson assembly reaction ===
 +
* add your purified PCR poducts and add water to reach the desired concentration as specified by your commerical kit or home-brew recipe.
 +
* 60<sup>o</sup>C for 1 hour
 +
** do in a thermocycler, and have it hold at 4<sup>o</sup>C forever afterward
 +
=== Transformation ===
=== Transformation ===
 +
* electroporation is the best method, as it can give you a very high efficiency
 +
* It is important to use a concentrated batch of electrocompetent cells.  When making your own, resuspend in 1/500th volume of the original growth medium.
 +
 +
=== Screening ===
 +
* If you plate a ton of biomass, look for colonies with skepticism.
 +
** If there is a lot of biomass, you might get a little growth of cells at the top of the plate as they are insulated from the antibiotic.  Restreak potential colonies to overcome this effect.
 +
*Use colony PCR to generate PCR fragments that will confirm your assembly.
 +
** Usually you will sequence across the whole insert and look for colonies that have an insert the length of your design.
 +
** I use [2X OneTaq http://www.neb.com/nebecomm/products/productM0486.asp] PCR mix for several reasons:
 +
*** It is cheap
 +
*** I know you can make a 1x mix (add the necessary water and primers) and use the mix after many freeze-thaw cycles.
 +
*** It has loading dye already so loading into agarose gels for observation is expedited.
 +
* To do colony PCR:
 +
** Decide how many colonies you want to screen.  Prepare a plate that is divided into numbered sections.    The colonies you select will be assigned these numbers.  Prepare a PCR strip (or strips) with the wells numbered and matching the colony numbers.
 +
*** I do 12 colonies because the agarose gel has enough lanes for this and ladder. 
 +
** After transformation, use a pipette tip to grab a single colony.
 +
** With this single colony:
 +
*** suck some up with the pipette
 +
*** deposit some on a section of an agarose plate with the appropriate antibiotic and put the remainder into the PCR well with the same number. 
 +
**** It is possible to overload it if you have really big colonies and suck up a lot of it with the pipette tip.
 +
*** Run the PCR with the correct extension temperature of the enzyme & the correct annealing temp for the primers.  (68oC for OneTaq. 55oC works for VF2 and VR primers)
 +
* Run the PCR products on a gel with ladder
 +
** We like Fermentas MassRuler
 +
*** The bands are sharp and the band sizes are intuitive.
=== Sequencing ===
=== Sequencing ===
 +
* Select 2-4 colonies for sequencing based on colony PCR
 +
* Sequence the seams of the Gibson assembly first.
 +
* Sequence the other regions, as it is possible a PCR error was introduced
 +
** Usually when an "error" is found, it was actually present on the template.
== Consolidated Version of Protocol ==
== Consolidated Version of Protocol ==
 +
Note: I have prepped a [https://docs.google.com/spreadsheet/ccc?key=0AlVxrZi130nMdHU3Tm1NSGN6Vlp2OEd1OXRpaUROQ3c#gid=24 spreadsheet template] that may make your first Gibson experience easier.  Anyone can view it, but I don't want people mistakenly changing the original, so I can send you a copy if you request one. -[[User:Janet B. Matsen|JM]]   
# Make a plasmid map of your design
# Make a plasmid map of your design
# Design Primers & generate annotated sequences of the bands you intend to create
# Design Primers & generate annotated sequences of the bands you intend to create
## primers should confer 40-100 bp of homology & be 60 bp long (in most cases)  
## primers should confer 40-100 bp of homology & be 60 bp long (in most cases)  
## 62<sup>o</sup>C < T<sub>m</sub> < 65<sup>o</sup>C as calculated by the [http://www.finnzymes.fi/tm_determination.html Finnzymes website]
## 62<sup>o</sup>C < T<sub>m</sub> < 65<sup>o</sup>C as calculated by the [http://www.finnzymes.fi/tm_determination.html Finnzymes website]
-
## * Check primers for cross dimers with Finnzyme's [http://www.google.com/url?q=http%3A%2F%2Fwww.finnzymes.fi%2Fjava_applets%2Fmultiple_primer_analyzer.html&sa=D&sntz=1&usg=AFQjCNGBTnwPK8yo48XqV5kq14dVTMt4sg multiple primer analyzer]
+
## Check primers for cross dimers with Finnzyme's [http://www.google.com/url?q=http%3A%2F%2Fwww.finnzymes.fi%2Fjava_applets%2Fmultiple_primer_analyzer.html&sa=D&sntz=1&usg=AFQjCNGBTnwPK8yo48XqV5kq14dVTMt4sg multiple primer analyzer]
## Make sure the reverse primer is reverse complemented!
## Make sure the reverse primer is reverse complemented!
# Double check primer design before ordering.  
# Double check primer design before ordering.  
-
## Blast your primers and templates with [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&QUERY=&SUBJECTS= blastn] and make sure they only anneal where you expect.
+
## Blast your primers and templates with [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&QUERY=&SUBJECTS= blastn] and make sure they only anneal where you expect. If there is a potential for mispriming with a high (>55<sup>o</sup>C) annealing temperature, consider trying to alter your design to prevent problems during PCR.  
## Blast the APE files for the expected PCR products against each other  
## Blast the APE files for the expected PCR products against each other  
# Generate PCR fragments  
# Generate PCR fragments  
Line 62: Line 113:
# Purify PCR fragments  
# Purify PCR fragments  
## Gel or sometimes PCR cleanup.
## Gel or sometimes PCR cleanup.
-
### Elute in 30 uL to obtain a concentrated product.  
+
### Elute in ~30 uL to obtain a concentrated product.  
## Measure DNA concentration with a nano drop
## Measure DNA concentration with a nano drop
# Plan Gibson Assembly reaction
# Plan Gibson Assembly reaction
## Use ~ 60 ng of backbone and stoichiometric quantities of insert(s)  
## Use ~ 60 ng of backbone and stoichiometric quantities of insert(s)  
# Transform
# Transform
-
## Electroporate 1 uL into a cloning strain.  Can do multiple electroporations and plate the cells together after they have grown out at 37<sup>o</sup>C.
+
## Electroporate 1 uL into a cloning strain.  Can do multiple electroporations and plate the cells together after they have grown out at 37<sup>o</sup>C.
 +
== If you get stuck ==
 +
=== At the assembly step ===
 +
* If you have short pieces, you can sew them together with overlap extension.  It is often easy to sew two pieces together if one is short (<1kb) or if both are < 2-4 kb.  Sewing together larger (~4kb) segments will probably cause you trouble.  See [[Lidstrom:Overlap_Extension_PCR|Overlap Extension PCR]]
== Examples ==
== Examples ==
Line 79: Line 133:
* Replacing short sections like ribosome binding sites
* Replacing short sections like ribosome binding sites
** primer will necessarily have homology in two places. **DRAW SKETCH**
** primer will necessarily have homology in two places. **DRAW SKETCH**
-
*
+
*'''HOMOLOGY'''
 +
** Causes problems during PCR and assembly.  Homology within a hundred or even a few hundred base pairs of the end can lead to recombination, as the exonuclease can be very fast.
 +
* toxic protein
 +
** if you are trying to clone in a toxic protein, your assembled plasmid may be too toxic to yield colonies
== Making your own Gibson mix ==
== Making your own Gibson mix ==

Revision as of 16:16, 22 March 2013

Back to Janet

Contents

Intro

  • What is it?
    • Important: the efficiency is low, however, you are selecting for complete/circularized products at the transformation step. Usually you will have no shortage of colonies to screen and sequence.
  • How does it differ from other cloning?
  • When should I use it?
  • Steps (concise)
    • Design oligos to yield 40 - 100 bp overlapping linear DNA segments
    • Purify (usually gel) the PCR products (or digest)
    • Use Gibson Assembly Mix
    • Transform
      • Electroporation is usually used to provide higher yield.

Other Resources

  • Guide by the creaters of Gibthon (software I haven't tried)
  • I don't like NEBuilder (web design tool):
    • It gave me overlaps of only 20 bp. I didn't see an option to request longer primers (to get more homology). When I changed the minimum to 60 bp it added more length to the priming portion of each primer, not the the homology-conferring portion. Strange...
    • It doesn't use backbone primers that confer homology. It only primes at the border. I have always used long primers that make homology to my insert. I am aware that this means I can't have re-usable backbone amplifications, but I feel it could only be advantageous for assembly. I am asking Rob Egbert if he thinks this matters much.

Protocol (descriptive version)

  • See below for consolidated version.

Prepare plasmid maps

  • Make a plasmid map of what your completed design should look like
    • This is key. You will want it for primer design, checking your primers, assessing sequencing reactions, etc. I use APE, open-source software. See my APE use page
    • Make sure each gene has a promoter, RBS, and stop codon.
    • If you aren't familiar with your sequences, make sure the sequence has no stop codons with the frame of reference it starts with. When downloading a sequence from somewhere like Genbank or getting a plasmid from somebody, this check should be done.
  • Make a plasmid map for each segment you will PCR amplify from a template
    • Include only the regions that will be present in your final assembly.
    • Do not include any homology regions initially. As you design your primers, make a new copy of each file that shows what the fragment will look like after priming as a PCR product.

Design primers

  • The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.
  • The annealing portion of the primer should have Tm between 62oC and 65oC as calculated by this Finnzymes website
    • This formula is applicable to Phusion DNApolymerase, the DNA polymerase used to form the DNA you will assemble.
  • Use cheap primers
    • If ordering with IDT, primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp reduces the length of the homolgy between adjacent DNA pieces in the assembly. Note: there are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***
  • Check primers for cross dimers with Finnzyme's multiple primer analyzer. If the annealing temperature of the primer dimer(s) is low, this will probably not be a problem during PCR.
  • Make sure the reverse primer is reverse complemented!

Double Check your Design

  • Blast your primers and templates with blastn and make sure they only anneal where you expect.
  • Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap. Make sure there is not extensive homology in other places.

Generate PCR fragments

  • I run each PCR at a few annealing temps and DMSO concentrations. See my sample spreadsheet.
  • Dpn1 can be added after the PCR is complete to degrade the template DNA. This will reduce the number of background colonies when you transform.
    • you will only get background if the antibiotic marker of the template is that of your design goal.
    • gel purification without doing DPN1 digestion usually is sufficient to greatly reduce background.
    • here is a sample result of background for a scenario where I used ~0.5 ng of template plasmid per 25 uL of PCR reaction to produce my backbone, then column purified (not gel purified!), and didn't do a DPN1 digestion. The pink colonies are the plasmid template carrying through the column purification, into the assembly reaction and transformation step.
      pink colonies in this transformation of a Gibson assembly are carry-through of template from the PCR amplification of the backbone
      pink colonies in this transformation of a Gibson assembly are carry-through of template from the PCR amplification of the backbone
  • Run a few uL of each PCR product on a gel to identify rxn conditions that yield a lot of product.

Purify PCR fragments

  • By default, you should gel purify your PCR bands. This will remove primer dimers, and undesired bands. Unfortunately, the column-based gel extraction kits have low efficiency. Elute in the buffer provided in the kit (presuming it is only 10 mM Tris, pH 8.5 & has no EDTA) to get the maximum amount of DNA back off the column.
    • Elute in 30 uL to provide a concentrated product.
  • You can do a PCR-cleanup instead to get higher yield *if* you run a few uL of the PCR product and it looks totally perfect. If you have ~ 100 uL of PCR product and the band is strong, I recommend gel purifying anyway. You will lose some, but the purity of your PCR product will be advantageous for assembly.
    • Using Dpn1 on the PCR product is good if you are going to do this, especially for anything that had template with the antibiotic resistance of your design goal, and if it is a backbone amplification.
    • Elute in 30 uL.
  • You will want ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are tolerable. If the band in your gel is strong and you have ~100 uL of PCR product, yields of ~ 50 ng/uL are more common.

Gibson assembly reaction

  • add your purified PCR poducts and add water to reach the desired concentration as specified by your commerical kit or home-brew recipe.
  • 60oC for 1 hour
    • do in a thermocycler, and have it hold at 4oC forever afterward

Transformation

  • electroporation is the best method, as it can give you a very high efficiency
  • It is important to use a concentrated batch of electrocompetent cells. When making your own, resuspend in 1/500th volume of the original growth medium.

Screening

  • If you plate a ton of biomass, look for colonies with skepticism.
    • If there is a lot of biomass, you might get a little growth of cells at the top of the plate as they are insulated from the antibiotic. Restreak potential colonies to overcome this effect.
  • Use colony PCR to generate PCR fragments that will confirm your assembly.
    • Usually you will sequence across the whole insert and look for colonies that have an insert the length of your design.
    • I use [2X OneTaq http://www.neb.com/nebecomm/products/productM0486.asp] PCR mix for several reasons:
      • It is cheap
      • I know you can make a 1x mix (add the necessary water and primers) and use the mix after many freeze-thaw cycles.
      • It has loading dye already so loading into agarose gels for observation is expedited.
  • To do colony PCR:
    • Decide how many colonies you want to screen. Prepare a plate that is divided into numbered sections. The colonies you select will be assigned these numbers. Prepare a PCR strip (or strips) with the wells numbered and matching the colony numbers.
      • I do 12 colonies because the agarose gel has enough lanes for this and ladder.
    • After transformation, use a pipette tip to grab a single colony.
    • With this single colony:
      • suck some up with the pipette
      • deposit some on a section of an agarose plate with the appropriate antibiotic and put the remainder into the PCR well with the same number.
        • It is possible to overload it if you have really big colonies and suck up a lot of it with the pipette tip.
      • Run the PCR with the correct extension temperature of the enzyme & the correct annealing temp for the primers. (68oC for OneTaq. 55oC works for VF2 and VR primers)
  • Run the PCR products on a gel with ladder
    • We like Fermentas MassRuler
      • The bands are sharp and the band sizes are intuitive.

Sequencing

  • Select 2-4 colonies for sequencing based on colony PCR
  • Sequence the seams of the Gibson assembly first.
  • Sequence the other regions, as it is possible a PCR error was introduced
    • Usually when an "error" is found, it was actually present on the template.

Consolidated Version of Protocol

Note: I have prepped a spreadsheet template that may make your first Gibson experience easier. Anyone can view it, but I don't want people mistakenly changing the original, so I can send you a copy if you request one. -JM

  1. Make a plasmid map of your design
  2. Design Primers & generate annotated sequences of the bands you intend to create
    1. primers should confer 40-100 bp of homology & be 60 bp long (in most cases)
    2. 62oC < Tm < 65oC as calculated by the Finnzymes website
    3. Check primers for cross dimers with Finnzyme's multiple primer analyzer
    4. Make sure the reverse primer is reverse complemented!
  3. Double check primer design before ordering.
    1. Blast your primers and templates with blastn and make sure they only anneal where you expect. If there is a potential for mispriming with a high (>55oC) annealing temperature, consider trying to alter your design to prevent problems during PCR.
    2. Blast the APE files for the expected PCR products against each other
  4. Generate PCR fragments
    1. Run each PCR with a few annealing temps and DMSO concentrations
    2. Check ~ 1.7 uL of each PCR producg on an 0.7% agarose gel and identify reaction conditions that gave product and don't have undesired bands.
    3. Optional: the good DNA can be treated with Dpn1
      1. Use ~ 1 uL per 50 uL PCR product to degrade unwanted template DNA
  5. Purify PCR fragments
    1. Gel or sometimes PCR cleanup.
      1. Elute in ~30 uL to obtain a concentrated product.
    2. Measure DNA concentration with a nano drop
  6. Plan Gibson Assembly reaction
    1. Use ~ 60 ng of backbone and stoichiometric quantities of insert(s)
  7. Transform
    1. Electroporate 1 uL into a cloning strain. Can do multiple electroporations and plate the cells together after they have grown out at 37oC.

If you get stuck

At the assembly step

  • If you have short pieces, you can sew them together with overlap extension. It is often easy to sew two pieces together if one is short (<1kb) or if both are < 2-4 kb. Sewing together larger (~4kb) segments will probably cause you trouble. See Overlap Extension PCR

Examples

  • Break up backbone if it is large (> 4kb??)
    • Only need 2 short primers to break it up: the homology is free.
  • you can chose where the seam is if you use longer oligos
  • RFP for backbone: don't screen red colonies!

Tricky Cases

  • Replacing short sections like ribosome binding sites
    • primer will necessarily have homology in two places. **DRAW SKETCH**
  • HOMOLOGY
    • Causes problems during PCR and assembly. Homology within a hundred or even a few hundred base pairs of the end can lead to recombination, as the exonuclease can be very fast.
  • toxic protein
    • if you are trying to clone in a toxic protein, your assembled plasmid may be too toxic to yield colonies

Making your own Gibson mix

  • Recipe
  • Tips:
    • Balancing the ratio of T5 & Phusion is mportant given the mechanism. The exonuclease is so concentrated relative to the desired concentration in the mix that it should be diluted 10X before use.
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