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Alternatively, copy and paste can be used to populate the Insert Fragment dialog. To copy the fragment to be inserted, open the file for the insert, select the restriction fragment, and click Edit → Copy Restriction Fragment.Then switch to the vector file, select the insertion site or the restriction fragment to be replaced, and click Edit → Paste. Welcome to Vector Database! Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. This is a free resource for the scientific community that is compiled by Addgene.

Associated Data

Supplementary Materials

Abstract

Multichange ISOthermal(MISO) mutagenesis is a new technique allowing simultaneous introduction ofmultiple site-directed mutations into plasmid DNA by leveraging two existingideas: QuikChange-style primers and one-step isothermal (ISO) assembly.Inversely partnering pairs of QuikChange primers results in robust, exponentialamplification of linear fragments of DNA encoding mutagenic yet homologous ends.These products are amenable to ISO assembly, which efficiently assembles theminto a circular, mutagenized plasmid. Because the technique relies on ISOassembly, MISO mutagenesis is additionally amenable to other relevant DNAmodifications such as insertions and deletions. Here we provide a detaileddescription of the MISO mutagenesis concept and highlight its versatility byapplying it to three experiments currently intractable with standardsite-directed mutagenesis approaches. MISO mutagenesis has the potential tobecome widely used for site-directed mutagenesis.

Site-directed mutagenesis (SDM) is one of the most frequently used techniques inmolecular biology. The QuikChange reaction, developed by Stratagene (La Jolla, CA), isthe standard approach for introducing point mutations into plasmids. Its widespread useto make specific coding changes in proteins has driven fundamental discoveries in thefields of genetics, biology, and biochemistry. QuikChange uses reverse complementarymutational primers to replicate a parent plasmid, introducing mutation(s) at the site ofprimer binding. The DNA replication step is a linear cyclic amplification reaction inwhich a pfu polymerase copies the entire plasmid, stopping uponreaching the primer’s 5′ end; the newly synthesized DNA is nicked andcannot serve as a template in later cycles but rather anneals to form nickeddouble-stranded, mutagenized molecules. Enzymatic digestion of methylated template DNAproduced in E. coli using DpnI reduces the background of wild typeparental molecules in the reaction.

Since its invention, only modest improvements to the QuikChange reaction havebeen proposed,^- and much inefficiency still exists in thissystem. First, while performed on a thermal cycler, DNA replication is linear notexponential. Second, strand displacement by the polymerase results in exponentialamplification of the plasmid and encodes a competing byproduct that cannot transformE. coli. Third, it is difficult to introduce mutations at more thanone location in any single reaction. Fourth, the size of the template is limitingbecause the method relies on replication of the entire plasmid. Fifth, because theentire plasmid must be copied, resequencing all nonselectable coding regions isobligatory. Sixth, background depletion by DpnI can be difficult because a large amountof DNA (50 ng) is recommended for the QuikChange reaction and hemimethylatedheteroduplex DNA is resistant to DpnI digestion.^, Seventh, becauseQuikChange primers are perfectly complementary, primer dimerization is highly favorable;long nick-bridging primers may minimize this and improve amplification. Finally, deletions and insertions largerthan a single codon are generally beyond the scope of the classic QuikChange reaction;however, modified QuikChange protocols attempt to address this shortcoming.

Herein we describe a simple and robust protocol for site directed mutagenesisthat overcomes all of the above challenges of the standard QuikChange reaction. We callour approach Multichange ISOthermal (MISO)mutagenesis since it is capable of introducing multiple DNA modifications in a singlereaction and incorporates a DNA assembly strategy named onestep isothermal (ISO)assembly. MISO mutagenesis isa completely different strategy from QuikChange; however, it still leverages the elegantdesign of QuikChange primers, which are reverse complementary sequences, usually 40 bpin length, that encode desired base changes centrally. In the simplest application ofMISO mutagenesis, two pairs of QuikChange primers are inversely partnered toexponentially amplify two linear, double-stranded PCR products (Figure 1a). The resulting PCR products encode the desiredmutations at each end and moreover share ~40 bp of terminal homology. Theone-step isothermal (ISO) reaction works by using a master mix of three enzymes toseamlessly assemble DNA pieces whose ends contain 30–40 base pairs ofoverlapping sequence (Figure 1b). Briefly, a5′ exonuclease chews back double-stranded DNA molecules to expose complementarysingle-stranded DNA overhangs. Homologous segments then specifically anneal. Next, apolymerase fills in the gapped molecules, and a ligase covalently seals nicks. Thus, PCRproducts with homologous ends, such as those generated through inverse partnering ofQuikChange primers, may be enzymatically joined in vitro using theone-step ISO assembly protocol to generate the desired mutagenized plasmid.

Overview of Multichange ISOthermal(MISO) mutagenesis. (a) QuikChange-style primer pairs (A, A′; B,B′) encode reverse complementary 40-nucleotide primers with a basesubstitution (star). Inverse partnering of primer pairs[A+B′] and[B+A′] in separate PCR reactions yieldsexponential amplification of two linear pieces of DNA with homologous ends.After template removal (DpnI digestion or gel purification), the mutagenizedplasmid is assembled using one-step isothermal assembly. (b) One-step isothermal assembly relieson the concerted action of three enzymes. A 5′ exonuclease chews backdouble-stranded DNA, exposing complementary single strands that anneal. Then apolymerase fills in the gaps, and a ligase seals the nick.

To demonstrate a robust capability for multi-site directed mutagenesis, we testedMISO mutagenesis with a set of 6 QuikChange primers encoding 8 base changes. Theseprimers were originally designed to incorporate eight lysine-to-arginine point mutationsinto a 6.5-kb plasmid using a combination of iterative QuikChange reactions and overlapextension PCR.^, We inversely partnered the six primer pairs(Figure 2a) to exponentially amplify sixdouble-stranded DNA fragments, ranging in size from 140 bp to 5.3 kb. The fragments weregel purified, subjected to one-step ISO assembly, and transformed into competentE. coli cells. As a control, one 140-bp fragment was omitted fromthe reaction, and the resulting assembly produced no colonies. Colony PCR reactions on96 individual transformants using two diagnostic primer pairs (Supplementary Figure 1a and b) revealedthat 92/96 clones had assembled correctly (Supplementary Figure 1c). Sequencing of 24correctly assembled constructs revealed that 100% contained all 8 desiredmutations. Thus, in a single round of experimentation we successfully generated alysine-free version of a protein of interest for assessment of post-translationalmodification status. These datasupport MISO mutagenesis as a tremendously improved strategy for multi-site directedmutagenesis.

Three applications of MISO mutagenesis. (a) Simultaneous introduction of eightpoint mutations into the mGOAT coding sequence. Six different pairs ofQuikChange-style primers were partnered (shown by colors) to generate six PCRproducts ranging in size from 140 bp to 5.3 kb and encoding eightlysine-to-arginine substitutions. One-step isothermal assembly reactionefficiently generated the desired lysine-free construct (see Supplementary Figure 1). (b)Introduction of a single point mutation into an existing 15.3-kb constructwithout vector segment amplification. QuikChange-style primers were partneredwith non-mutagenic primers complementary to plasmid ends to generate two PCRproducts (200 bp, 800 bp). Separately, pLD401 was digested with AscI and BstBI,and the large vector fragment was gel purified. The three DNA fragments, withhomologous ends, were subjected to one-step isothermal assembly to construct themutagenized plasmid. Only the region of the plasmid produced by PCR requiredconfirmatory resequencing. (c) Simultaneous introduction of a base substitution,deletion, and insertion into yeast shuttle vectors. One standard set ofQuikChange primers (starred) plus three other primer pairs were partnered (shownby color) to generate four overlapping PCR products. One-step ISO assemblyallowed deletion of two BsmBI sites from a noncoding region, recoding of oneBsaI site in the bla gene, and insertion of a BsaI-flanked RFPgene to generate a new cloning site. [Open gray arrows = codingsequence; stars = point mutations; scissors = unique restrictionenzyme sites; orange circles = undesirable restriction enzymerecognition sites; dashed lines = deleted region.]

Another limitation of QuikChange is the size of the template plasmid. As thereaction mandates replication of the entire construct, the upper limit for QuikChange is~7–10 kb. Further, many large plasmids carry a significant fraction ofcoding region, and any that cannot be functionally validated following QuikChange mustbe entirely resequenced to verify accuracy. Here we demonstrate additional versatilityof MISO mutagenesis to overcome these two issues by coupling MISO mutagenesis with atraditional restriction digestion. To introduce a single point mutation into a 15.3-kbplasmid of which ~9 kb encodes protein sequence (Figure 2b), we identifiedunique restriction enzymes sites flanking the desired base substitution by 200 bp and800 bp and designed primers to anneal beyond these boundaries. In individual PCRreactions, we inversely partnered these primers with two QuikChange-style primersencoding the mutation. Separately, the backbone was digested with the appropriaterestriction enzymes, and the three fragments were gel purified, thereby quicklygenerating three overlapping pieces of DNA amenable to one-step ISO assembly. Weconfirmed introduction of the mutation in five out of five unique transformants bysequencing. Further, one of these constructs was sequence verified at both overlappingjunctions. Here, not only did MISO mutagenesis allow efficient installation of themutation of interest, it also greatly reduced the amount of sequence validationrequired.

DNA modifications of biological relevance are not limited to base substitutions.Rather, the introduction of insertions and deletions into plasmids is often desirable,for instance, to generate fusion proteins, co-expression systems, or to delete proteindomains. To this end, we next used MISO mutagenesis to couple the introduction of apoint mutation with simultaneous deletion of a DNA segment and insertion of a 1-kbsequence into a series of yeast shuttle vectors (Figure2c). Briefly, we neededto recode a single BsaI site within the bla gene, remove two BsmBIsites from a noncoding region of the vector backbone, and construct a new cloning sitewith BsaI sites flanking a red fluorescent protein (RFP) to generate new host plasmidsamenable to Golden Gate assembly. Wedesigned QuikChange primers for BsaI recoding plus three additional pairs of primerswith overlapping overhangs (Figure 2c), which wereused to exponentially amplify four fragments with homologous ends. Gel purification ofall amplification fragments prior to one-step ISO assembly yielded ~99%red colonies, and 20 were miniprepped and the assembly tested by restriction digestswith BsaI and BsmBI (SupplementaryFigure 2). We discovered that 19/20 clones yielded the expected digestionpattern; the single incorrect clone derived from an assembly error in which oneRFP-flanking BsaI site was not intact (Supplementary Figure 2). This highlights that one-step assembly reactionsare prone to mis-assemblies and junctions must be sequence verified. Thus, afterfunctional verification by restriction digestion, we further sequenced the BsaI-RFPjunctions of 8 correctly assembled clones to verify elements that could not beinterrogated by digest; no undesired mutations were found. Taken together, theapplication of MISO mutagenesis presents a simple strategy for making many types ofbiologically relevant DNA modifications in a single round of experimentation.

MISO mutagenesis overcomes many specific technical problems associated withtraditional QuikChange mutagenesis. Since primers are inversely partnered in separatereactions, the problem of primer dimerization is circumvented. Further, MISO mutagenesisaffords exponential rather than linear amplification, thus allowing easy verification ofproduct generation by gel electrophoresis. Background is reduced almost completelythrough template removal by gel purification and/or DpnI digestion. Like QuikChange,however, MISO mutagenesis is limited by DNA sequences that are challenging to amplify byPCR, or may be toxic, unstable, or otherwise not tolerated in bacteria. The error rateassociated with oligonucleotide synthesis is another problem common to both approaches.Limitations specific to MISO mutagenesis derive largely from the ISO-assembly step.First, as demonstrated here, mis-assembly errors can occur during one-step ISO assembly,and it is likely that mis-assemblies will be exacerbated by repetitive or GC-richsequences in homologous regions. It is possible that implementation of alternativeenzymatic assembly strategies may overcome some types of assembly errors.^, Second, the introduction of mutations that are relatively closetogether (e.g., 50–80 bp) may be difficult to achieve using MISO mutagenesis, asthis would require exceedingly long complementary mutagenic primers to encode bothmutations or alternatively generating a very short PCR product. Thus, MISO mutagenesisis likely best applied when desired mutations are close enough to be encoded together onone primer or distant enough to generate a reasonably sized PCR product using twomutagenic primers. Finally, the number of pieces that can be put together by one-stepISO assembly defines the upper limit of DNA modifications introduced during a singleround of MISO mutagenesis. Of course, most of these limitations can be overcome byperforming MISO twice.

Ape Plasmid Editor Manual Software

Overall, we believe that MISO mutagenesis is a versatile and efficient strategyfor making the most common types of DNA sequence modifications in plasmids. Thisapproach is accessible and cost-effective for all laboratories as it seamlesslyincorporates QuikChange-style primers, which are both familiar and ubiquitous inmolecular biology, and requires no expensive primer purification. Implementation of theone-step ISO assembly protocol is also straightforward as step-by-step instructions toprepare the three enzyme reaction mixture have been comprehensively detailed. Alternatively, kits with all therequired reagents preassembled are available from NEB. Thus, MISO mutagenesis representsan excellent solution for laboratories that infrequently perform SDM in plasmid DNA tothose where it is routine. Notably, single mutations can also be installed using MISOmutagenesis by inversely partnering QuikChange-style primers with a second pair ofnon-mutagenic primers or by employing the approach outlined in (Figure 2b). Indeed, we have used this approach to successfullysalvage failed QuikChange reactions (data not shown).

METHODS

Primer Design

Primers used in this study are listed in Supplementary Table 1. Formutagenic primers, 40 nucleotide exact reverse complementary sequences weredesigned with the base substitution placed centrally, as per the QuikChangemanual (Stratagene). In the case of insertions/deletions, the desired constructwas assembled in silico using the free plasmid editor ApE(http://biologylabs.utah.edu/jorgensen/wayned/ape/), keepingtrack of the junction locations. Primers were then designed to consist of twoparts: an annealing sequence (20–30 nt, T_m ≈ 55°C) and an ‘overhanging’ sequence to generate thehomologous region. In our hands, the minimal homologous region for ISO assemblyis ~30 bp, although 40 bp is recommended,^,and we have succeeded in assembling fragments that overlap by as much as 200 bp.As an example, the primers used to generate Figure2c are diagramed in Supplementary Figure 3. In all cases, overlapping regions wereconfirmed to be unique to each assembly reaction.

PCR Amplification of DNA Fragments

Phusion Polymerase (NEB, F530L) was used to generate all PCR productsdescribed here, although any high fidelity polymerase is appropriate for use.PCR reactions were prepared as follows: 5–10 ng template DNA, 200μM concentration of each dNTP (Takara, 4030), 0.2μM concentration of each primer (Supp. Table 1), 1x Phusion HFbuffer, 0.02 U/μL Phusion DNA polymerase in a finalvolume of 50 μL. Applied Biosystem Veriti 96-WellThermal Cyclers were used for amplifications with an extension time of 30 s/kb.PCR products were either gel purified using the Zymoclean Gel DNA Recovery Kit(Zymo Research, D4002) as per the manufacturer’s instructions orpurified using the DNA Clean & Concentrator kit (Zymo Research, D4003)with or without DpnI treatment (NEB, R0176) for 1 h at 37 °C.

One-Step ISO Assembly

One-step ISO assembly reagents (5X ISO Buffer and Reaction Master Mix)are described in detail elsewhere. PCR products were combined in equimolar amounts in 5μL and mixed with 15 μL ofReaction Master Mix by gentle tapping. One-step isothermal assembly wasperformed at 50 °C in a preheated PCR block for 30 min, and 2μL of each assembly reaction was transformed into50 μL of competent DH5α E.coli cells.

Ape Plasmid Software

Supplementary Material

Acknowledgments

This work was supported in part by a National Science Foundation (grant MCB-1026068)and a DARPA contract (N66001-12-C-4020) to J.D.B.

ABBREVIATIONS

MISO	multichange isothermal
ISO assembly	one-step isothermal assembly
PCR	polymerase chain reaction
DNA	DNA
nt	nucleotide
bp	base pair

Footnotes

Author Contributions

L.A.M., Y.C., and M.T. are co-first authors of this work and wrote themanuscript. J.C., L.A.M., and M.T. created the figures. L.A.M., J.C., A.M.N.,Y.C., L.D., and M.T. contributed experimental data. The work was performed inthe lab of J.D.B.

The authors declare no competing financial interest.

Supporting Information

Supplementary figures andtable. This material is available free of charge via the Internet athttp://pubs.acs.org.

References

Here are optimized methods and tips from the Chien lab, adopted after trial and error, which work consistently in our hands. See the Invitrogen multisite Gateway manual for all of the basic information necessary to understand and perform Gateway recombination reactions. See also the Gateway tips on the Lawson lab website.

Please report problems or questions on the Tol2kit blog.

As a first test, we suggest that you grow up entry clones and perform a test LR reaction with pDestTol2pA2 or pDestTol2CG2 to make an expression clone such as bactin2:EGFP-pA.

• 3BP Reactions
• 4LR reactions
• 5Assembling sequences for expression clones
• 6Injections for transgenesis

Growing up clones

Entry clones are kanamycin-resistant and can be transformed and grown in any standard E. coli strain. For a single lab's use, minipreps (e.g. using Qiagen miniprep columns) are sufficient for the entry vectors; you will use very little DNA for each LR reaction. If you run low, you can always just miniprep again.

However, note that destination vectors have an ampicillin resistance gene in the backbone, donor vectors have a kanamycin resistance gene in the backbone, and both have a ccdB suicide gene and a chloramphenicol resistance gene in the 'gate' (the ccdB provides negative selection during the BP or LR reaction). Therefore these clones must be grown in ampicillin/chloramphenicol or kanamycin/chloramphenicol, in ccdB-tolerant cells (available from Invitrogen). In addition, we have found that certain destination vectors and donor vectors are prone to recombination or mutation, so at a minimum you should test each DNA prep by careful restriction analysis. We have also found that while propagating these vectors, it is crucial to pour plates of the desired antibiotic resistance; it is not sufficient to pipet chloramphenicol solution onto a pre-poured ampicillin or kanamycin plate.

For donor vectors and destination vectors, which you will use repeatedly and which are tricky to grow up correctly, we recommend that you grow at least a midiprep or maxiprep scale.

We use the following antibiotic concentrations: ampicillin (100 ug/ml), kanamycin (50 ug/ml), and chloramphenicol (30 ug/ml), both for selection on plates as well as in liquid culture.

What you will need

Here are the specific reagents required for working with the Tol2Kit, including Invitrogen catalog numbers. Where more than one catalog number is listed, this reflects the different sizes available.

BP Clonase II Enzyme Mix (11789-020, 11789-100): for generating entry clones. Note: unlike BP Clonase, which had a separate buffer, BP Clonase II includes the buffer in the enzyme mix.

LR Clonase II Plus Enzyme Mix (12538-120, 12538-200): for generating expression constructs via the multi-site reaction. Note: LR Clonase II (no Plus) is a different enzyme, to be used for 'classic' (non-multisite) Gateway reactions. Note: make sure to store LR Clonase II Plus at -80 degrees, as it seems to be especially labile. (We usually also store the BP Clonase II and of course the One Shot cells at -80 degrees.)

One Shot ccdB Survival Competent Cells (C7510-03): for propagation of empty donor and destination vectors.

One Shot TOP10 Chemically Competent E. coli (C4040-10, C4040-03, C4040-06): for transformation of LR reactions. Note: do NOT use One Shot TOP10F' cells; these will not show a difference in clear and opaque colonies (see below).

pDONR221 (12536-017): empty middle entry vector for generation of new middle clones.

pDONR P4-P1R and pDONR P2R-P3 (12537-023): empty 5' and 3' donor vectors for generation of new 5' and 3' entry clones. Note: the catalog number provided here is for the MultiSite Gateway Three-Fragment Vector Construction Kit; it includes pDONR221 as well as a destination vector (pDest R4-R3). This appears to be the only way to purchase the empty donor vectors at this point.

Note that the Tol2kit is based on the original three-part multisite Gateway system (as described in the version D manual), in which destination vectors use attR4-R3 sites, not the new Multisite Gateway Pro system, in which all the destination vectors use attR1-R2 sites. While the donor, entry, and destination vectors are incompatible with the Pro system (different sets of att sites are used), the BP and LR enzyme mixes are still the same.

BP Reactions

Donor Vectors

The Invitrogen multisite Gateway manual describes and explains the donor vectors in detail. The one extra technical note is that the 5' donor vector (pDONR P4-P1R) can be tricky to propagate due to self-recombination. Based on advice from the Lawson lab, we now use their 5' donor vector propagation protocol.

PCR Amplification of DNA

Primers for PCR are designed as described in the multisite Gateway Manual. This results in primers that are quite long (regularly >50 bases), but we have not had difficulty performing PCR with these primers. This list of att site sequences may be useful.

We have used two different polymerases for PCR: Tth (GeneAmp XL PCR Kit; Applied Biosystems) and Phusion (NEB). Both are proofreading polymerases that can amplify long pieces of DNA, although for particularly difficult and/or long promoters, Phusion has worked better. For each, PCR was performed in a 50 ul reaction.

Purification of PCR products

The entire PCR reaction (50 ul) is loaded onto an agarose gel. The appropriate band is excised and DNA purification performed using the Qiagen Qiaquick Gel Extraction Kit (for DNA fragments <10 kb; for larger fragments, use the QIAEX Gel Extraction Kit). Elute the DNA in 30 ul (the smallest recommended volume). The concentration of DNA is calculated using a spectrophotometer; the DNA will be quite dilute and not terribly clean (usually between 10-80 ng/ul, and OD 260/280 ~1.4-1.6).

Do not let the gel-purified DNA sit in the freezer for too long before using in the recombination reaction. In practice, we go straight into the recombination reaction; a better stopping point is to freeze either the entire PCR reaction or the gel slice before purification. We have found that storing the DNA in the freezer for even a couple of days decreases the efficiency of recombination.

BP Recombination Reactions

The recombination reaction is performed as described in the Invitrogen Multi-Site Gateway Manual. An equimolar amount of the appropriate donor vector and purified PCR product (commonly 50-100 femtomoles) are combined with TE and BP Clonase II enzyme mix in a final volume of 10 ul. This reaction is usually allowed to incubate overnight at room temperation, however, we have found (as the manual also suggests) that as little as 2 hours can be enough. This reaction almost always works well. Note that the suggested reaction volume of 10 ul is the 'full' reaction suggested by the Invitrogen manual. Other labs have moved to setting up half-reactions (5 ul final volume); this works as well.

Transformation, Plasmid Prep, and Diagnostic Digests

The BP reaction is treated with Proteinase K and transformed. Typically, 2 ul of the 10 ul reaction is sufficient. The Invitrogen Manual recommends OneShot TOP10 cells, but cells of this high competence are not necessary. Subcloning efficiency cells and also homemade competent cells have worked well, yielding hundreds to thousands of colonies per plate. We typically pick 4 colonies for minipreps, and these are almost always the correct clone. Check by restriction, and then by sequencing for PCR errors. For restriction tests, we try to pick enzymes that do not cut in the att sites. PvuII is often useful, as are EcoRV and HindIII.

Note: the clear/opaque difference in colonies applies only to transformants from the LR reaction, not this (the BP) reaction.

LR reactions

Recombination Reactions

The recombination reaction is performed with slight modifications from the protocol in the Invitrogen Multi-Site Gateway manual. Equimolar amounts of entry vectors (5', middle, and 3') and destination vector are combined with LR Clonase II Plus enzyme mix. Note that unlike the earlier version (LR Clonase Plus), there is no separate buffer (it comes premixed with the enzyme). We standardly set up reactions with 20 femtomoles of each vector in a 10 ul reaction. The original manual has a protocol for a 20 ul reaction, however, this enzyme mix (LR Clonase II Plus) comes with a protocol for a 10 ul reaction. Other labs have found that half reactions (5 ul) work as well.

We always allow this reaction to go overnight at room temperature. The reaction tends to be less efficient than the BP reaction, likely because of the number of components involved.

Transformation, Plasmid Prep, and Diagnostic Digests

As with the BP reaction, the LR reaction is treated with Proteinase K and then transformed. We typically transform 3 ul of the 10 ul reaction, using Invitrogen OneShot TOP10 cells. Because this reaction is less efficient than the BP reaction, cells of this high competence are necessary. The protocol for these cells recommends shaking at 37 degrees for one hour after heat shock. Instead, we generally shake for 1.5 hours, just to give the cells more time to grow before selection. We then plate all 300 ul onto the LB/amp plate. Particular LR recombination reactions can be less efficient than others (see below), and we believe that giving the culture one more doubling time will increase the chance that the correct clone can be isolated.

This reaction is plated onto ampicillin plates; carbenicillin works as well. We typically find hundreds of colonies per plate. We do not plate the reaction before 3 pm, as satellite colonies can be a significant problem, obscuring the results of the reaction. Plates are removed from the 37 degree incubator first thing the next morning; this provides the best chance to distinguish clear from opaque colonies. If it is difficult to tell clear from opaque, looking at the plate in front of a dark background (we use a black refrigerator) will help. The image below shows examples of clear and opaque colonies on the same plate.

Plates can be left at room temperature until clear colonies are picked in the afternoon. We have found that clear colonies contain the correct clone >99% of the time, while opaque colonies never contain the correct clone. A reaction that has worked well will have a clear to opaque colony ratio of at least 3:1. However, as long as clear colonies can be identified, the correct clone will be isolated. As with the BP reaction, clones are tested via restriction digest; again, we generally avoid enzymes that cut within the att sites. PvuII has been very useful for this.

Factors Affecting Reaction Efficiency

Certain entry vectors seem to be less efficiently recombined in the LR recombination reaction. The lower efficiency of the reaction will be conveyed by a lower number of transformants, as well as a lower ratio of clear to opaque colonies. The major factor leading to lower recombination efficiency appears to be size of the insert. Both particularly short and extremely long DNA fragments can be tricky. Short is defined as less than 200 bases (e.g. p3E-polyA in the Tol2Kit), and long is defined as greater than 10 kb. Although these reactions work less efficiently than others, we have not defined a lower limit for fragment length. For example, p5E-Fse-Asc has an insert of 59 bases; this will still work in recombination reactions. On the other hand, entry vector fragments of greater than 10 kb have been difficult to work with; it is not clear if this reflects something about the recombination reaction or the specific DNA insert.

Assembling sequences for expression clones

Each of the 4 sets of att sites has a core sequence, e.g. 'attB4/L4/R4_shared', which is shared between the attL, R, B, and P sites (see sequences here). This means that adjacent clones used to build an expression clone will have at least a 15 bp overlap at the ends. This overlap can be used to predict the sequence of the expression clone, for instance to pick diagnostic restriction digests.

Ape Plasmid Editor Guide

by hand or with Sequencher

Here are relevant sequences (entry clone inserts, destination clone ends) and a detailed description of how to build expression clone sequences using a simple sequence assembly program like Sequencher.

using ApE

12/18/07: We have now started using Wayne Davis' really lovely shareware program [ApE] ('A plasmid Editor'), which can calculate Gateway recombinations for you:

• Open sequences for the three entry clones and destination clone (e.g. using the Genbank-format sequences provided on the wiki). (Make sure that all sequences are marked as circular, not linear.)
• Select Tool>Recombination Tool..., and select the multisite Gateway prototype.
• Hey presto, you have your expression clone sequence.

ApE does lots of other things too--think DNA Strider on steroids. Thanks Wayne!

sequence deviations

When sequencing entry clones, you may occasionally notice (as we have) a single base in an att site differing from that given by Invitrogen. Apparently, their documented sequence is not base-perfect. We will list these differences here as we notice them.

C>A change (shown in lowercase here) in the attL1 and attP1 sequences:

Injections for transgenesis

Preparation of capped transposase RNA

pCS2FA-transposase is linearized using NotI and purified using the Qiagen PCR Purification Kit. In vitro transcription is carried out using the Ambion mMessage mMachine SP6 Kit (catalog #1340); 2 ug linearized DNA is used in each transcription reaction. In vitro transcribed RNA is purified using the Qiagen RNeasy Mini Kit, and subsequently ethanol precipitated and resuspended in a final volume of 20 ul. The RNA concentration is then quantified using a spectrophotometer, and run on a gel to confirm its integrity. We generally get 20 ul of approximately 1 ug/ul capped RNA from each reaction.

Injections

Injections are performed at the 1-cell stage. It is crucial to inject the nucleic acids into the cell (not the yolk); this most increases the chance of early integration.

Spotting DNA for distribution

For distribution, we usually dilute maxiprep DNA to 250 ng/ul in 0.025% bromphenol blue (using a 10x stock of 0.25% BPB in TE), then spot 2 ul per construct onto Whatman paper (final amount 500 ng). In cases where the maxiprep concentration was low, we have spotted as little as 125 ng.