Janet B. Matsen:Guide to Gibson Assembly
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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)
- 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.
- Screen w/ colony PCR
- 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.
- If ordering with IDT, primers should usually be 60 bp if you are encoding homology.
- 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.
- 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:
- 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.
- This is to avoid template carry-through.
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.
- If your product is specific and doesn't need to be gel purified: (only needs PCR cleanup)
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.
- Desired outcomes:
- Column purifying 30uL of a strong PCR band should yield ~40 uL of ~30-50 ng/uL product.
- Gel purifying ~100 uL of PCR product usually yield ~ 50 ng/uL.
- You will want ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are usually fine.
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.
- Use 1-2 uL of Gibson assembly if it isn't desalted.
- 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)
- 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.
- Run the PCR products on a gel with ladder
- We like Fermentas MassRuler
- The bands are sharp and the band sizes are intuitive.
- We like Fermentas MassRuler
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
- Make a plasmid map of your design
- 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)
- 62oC < Tm < 65oC as calculated by the Finnzymes website
- Check primers for cross dimers with Finnzyme's multiple primer analyzer
- Make sure the reverse primer is reverse complemented!
- Double check primer design before ordering.
- 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.
- Blast the APE files for the expected PCR products against each other
- Generate PCR fragments
- Run each PCR with a few annealing temps and DMSO concentrations
- 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.
- Optional: the good DNA can be treated with Dpn1
- Use ~ 1 uL per 50 uL PCR product to degrade unwanted template DNA
- Purify PCR fragments
- Gel or sometimes PCR cleanup.
- Elute in ~30 uL to obtain a concentrated product.
- Measure DNA concentration with a nano drop
- Gel or sometimes PCR cleanup.
- Plan Gibson Assembly reaction
- Use ~ 60 ng of backbone and stoichiometric quantities of insert(s)
- Transform
- 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.