Janet B. Matsen:Guide to Gibson Assembly

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=== Purify PCR fragments ===
=== Purify PCR fragments ===
* The best way to purify PCR products is a simple column cleanup.  We use the Qiagen PCR cleanup kit, and elute in water.  
* The best way to purify PCR products is a simple column cleanup.  We use the Qiagen PCR cleanup kit, and elute in water.  
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** This usually requires your PCRs were very specific to the band size you wanted.  This is why PCR primers are done with melting temperatures of 70oC: doing annealing at a high temp (67-70<sup>o</sup>C) is the most likely way to give you the desired PCR amplification.  You need to have checked ~2-3uL of your product on an agarose gel to make sure your PCR was specific to your goals.   
+
** This usually requires your PCRs were very specific to the band size you wanted.  This is why PCR primers are done with melting temperatures of 70<sup>o</sup>C: doing annealing at a high temp (67-70<sup>o</sup>C) is the most likely way to give you the desired PCR amplification.  You need to have checked ~2-3uL of your product on an agarose gel to make sure your PCR was specific to your goals.   
* You can also gel purify your PCR bands, but you lose a LOT of product, and the product is lower quality.   
* You can also gel purify your PCR bands, but you lose a LOT of product, and the product is lower quality.   
** This will remove primer dimers, and undesired bands.  Unfortunately, the column-based gel extraction kits have extremely low efficiency.  You can elute in water or the buffer provided by the kit (presuming it is only 10 mM Tris, pH 8.5 & has no EDTA), but I always used water.   
** This will remove primer dimers, and undesired bands.  Unfortunately, the column-based gel extraction kits have extremely low efficiency.  You can elute in water or the buffer provided by the kit (presuming it is only 10 mM Tris, pH 8.5 & has no EDTA), but I always used water.   

Revision as of 09:41, 14 October 2013

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Contents

Intro

  • Gibson assembly allows for seamless cloning, pretty easily.
  • The efficiency of assembly is low (like for restriction cloning), however, you are selecting for complete/circularized products at the transformation step. For smaller plasmids (6kb or less) with genes that aren't horribly toxic, you should have no shortage of colonies to screen and sequence.
  • It can be used for site directed mutagenesis: NEB guide

Steps (concise)

  1. Design oligos to yield 40 - 100 bp overlapping linear DNA segments
  2. Purify (usually gel) the PCR products (or digest)
  3. Use Gibson Assembly Mix
  4. Transform
    1. Electroporation is usually used to provide higher yield.
  5. Screen w/ colony PCR
  6. Sequence 1-3 candidates

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.
    • Mostly, this means copying from other plasmid sequences and pasting into a new plasmid file.
      • However, you can add shorter items like promoters and ribosome binding sites by coding for them in your primers. Also, you can amplify genomic DNA for insertion.
  • Double check your design
    • Make sure each gene has a promoter, RBS, and stop codon if desired.
    • 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 (optional)
    • Do include overlap generated by the primers.

Design primers

  • I always use primers with Tm ~70oC for the annealing region 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.
  • Make sure the reverse primers you are ordering are in fact reverse complemented.
  • Use cheap 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.
    • You usually only need one of the two primers to confer homology. If you use an 18-30 bp primer for one edge of a seam, and the other primer is 60 bp (including binding and homology), that is usually enough overlap.
    • Keep in mind the pricing structure from the oligo company you use.
      • If ordering with IDT, primers should usually 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.
      • Sometimes you need a longer (say 90bp) primer to add promoters/RBSs, or additions to a coding sequence. These primers work fine with the default purification; don't pay more for additional purification just because they are long.
  • Optional: 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.
    • I don't do this any more.

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

Find optimal conditions

  • 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.
  • Run a few uL (~4uL) of each PCR product on a gel to identify rxn conditions that yield a lot of product. Look for conditions that make a lot of your product, and ideally no other undesirable products.
    • Using Tm = 70oC (not lower) reduces the probability of unspecific bands greatly.
  • 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.
    • This is to avoid template carry-through.
      • If the templates for your PCRs are Kanamycin vectors, and you are building a Kanamycin vector then some fraction of your transformants will just be cells with the template plasmid(s) carried through. This needs to be kept in mind later at the screening step.
    • You can put 1/2-1 uL in your PCR product is complete; there is no need to modify the buffer first.
    • You will only get background if the antibiotic marker of the template is that of your design goal.
      • If you have a fragment from an Amp plasmid, and are building a Kanamycin vector, there is no need to add Dpn1.
    • 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

Make enough to purify and assemble

  • Run purification scale reactions to make DNA for assembly
    • If your product is specific and doesn't need to be gel purified: (only needs PCR cleanup)
      • 20uL of a strongly amplified insert is plenty. Do a bit more if it is the backbone.
    • If your product is co-amplified with other undesirable products and will need to be gel purified:
      • run more like 60-120 uL, depending on how bad the byproducts are.

Purify PCR fragments

  • The best way to purify PCR products is a simple column cleanup. We use the Qiagen PCR cleanup kit, and elute in water.
    • This usually requires your PCRs were very specific to the band size you wanted. This is why PCR primers are done with melting temperatures of 70oC: doing annealing at a high temp (67-70oC) is the most likely way to give you the desired PCR amplification. You need to have checked ~2-3uL of your product on an agarose gel to make sure your PCR was specific to your goals.
  • You can also gel purify your PCR bands, but you lose a LOT of product, and the product is lower quality.
    • This will remove primer dimers, and undesired bands. Unfortunately, the column-based gel extraction kits have extremely low efficiency. You can elute in water or the buffer provided by the kit (presuming it is only 10 mM Tris, pH 8.5 & has no EDTA), but I always used water.
    • Elute in 30 uL (not 50 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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