Janet B. Matsen:Guide to Gibson Assembly

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=== At the assembly step ===
=== 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]]
* 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]]
 +
** You are more likely to get PCR errors incorporated if you use this method.
 +
* You can make two assemblies that are each closer to your design goal, and reassemble them into the desired final product.
 +
** This is handy when your design is large (9 or more kilobases) or your genes are toxic.
== Examples ==
== Examples ==

Revision as of 21:03, 3 October 2013

Back to Janet

Contents

THIS ARICLE IS CURRENTLY INCOMPLETE AND A BIT OUT OF DATE

- JM 9/2013

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)
  • NEBuilder: A web-based primer design tool

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 comments in Tips & Tricks.
    • 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 in frame with the start.
  • Make a plasmid map (e.g. APE file) for each segment you will PCR amplify from a template
    • Include only the regions that will be present in your final assembly.
  • Optional: make plasmid maps for each piece you are making in PCR
    • Do include overlap generated by the 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.
    • Usually having a primer that overlaps just one segment in a joint is sufficient. Use one 60bp primer that confers homology to the adjacent segment and one 20-30bp primer for the other piece that does not confer homology.
  • I always use primers with Tm ~70oC when available. Tm values should always be calculated by the 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

  • Make sure the forward primers and reverse primers you are ordering match the intended direction.
    • This is an easy mistake to make.
  • Fill out a table like the picture below so you have an explicit record of the assembly.
    • Start a record keeping spreadsheet that has the primers, Tm values, template used, and expected bp.  Access to spreadsheet depicted: here
      Start a record keeping spreadsheet that has the primers, Tm values, template used, and expected bp. Access to spreadsheet depicted: here
    • You can reference these cells when you plan out PCR reactions.
  • You can blast your primers and templates with blastn to make sure they only anneal where you expect if you aren't super familiar with your sequences.
  • You can blast the APE files for the expected PCR products against each other to make sure they have sufficient overlap.

Generate PCR fragments

  • I run each PCR at a few annealing temps and DMSO concentrations. Example below:
    • Example of test-scale DNA synthesis batches.  Spreadsheet: here.
      Example of test-scale DNA synthesis batches. Spreadsheet: here.
    • DMSO can be important, especially if you are amplifying DNA from the genome of whole bacterial cells. The DMSO likely disrupts the membrane enough to allow the polymerase to work.
  • 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. JM uses Top10, but hasn't really tried other strains.
    • Use 1-2 uL of Gibson assembly if it isn't desalted.
      • If you want to use more, do a PCR cleanup desalt to remove salts & prevent arcing.
      • We use Millipore desalting paper, item #VSWP01300. Put the whole assembly on top of a filter that is floating on top of water. Leave for 1 hour. You can transform the whole reaction after this if you wish. A simple assembly (2 pieces, normal to small backbone & normal insert size) should give a lawn.
  • It is important to use electrocompetent cells that are SUPER viscous. There should be only enough 10% glycerol in the mix to allow pipetting. If the viscous cell suspension gets a little bit stuck while you are pipetting, you are around the right viscosity.

Screening

  • 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.
      • Alternately, you can make a primer/water mix that you aliquot into each PCR tube. After you add cells, add the green 2X mix and run in the thermocycler.
      • 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
    • You are more likely to get PCR errors incorporated if you use this method.
  • You can make two assemblies that are each closer to your design goal, and reassemble them into the desired final product.
    • This is handy when your design is large (9 or more kilobases) or your genes are toxic.

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

  • We used to make our own before New England Biolabs started selling it, but ours gives ~10x less colonies so we no longer make it.
  • 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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