Bp Clonase Manual

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So my reaction is not working and I am not able to get any colonies after the transformation of the reaction. Does anybody have any suggestions for an efficient BP reaction. Or its better to change primers. BP Clonase enzyme Gateway-Cloning Cloning Vector Caenorhabditis elegans Share Facebook Twitter LinkedIn Reddit All Answers (1) 15th Jan, 2014 Tine Horning Morthorst Aarhus University It is different which nucleotides I have had at that position. I use the following databses to design my primers for Gateway and then just ajust them for matching annealing temperature. ORF-Entry: Promoter-Entry: This works just fine for me. Besides that, when doing the BP reaction (and the LR reaction as well) I always incubate over night at 25 C. It gives higher yield in colonies. Hope you get it to work. Cite Can you help by adding an answer. Answer Add your answer Similar questions and discussions Best Amplitude value for sonication to E.coli BL21 (DE3)? Question 5 answers Asked 20th Jun, 2018 Abir Chakraborty Dear All, Sonication is a vital step in protein purification and over sonication can definitely damage the secondary structure of the protein(correct me if I am wrong). What is the best Hz value for sonication. Is Amplitude different from Hz.I have used two types of instruments: one only shows Amplitude value,other shows both. View Does anybody have experience in DAPI staining of Arabidopsis protoplasts. Question 11 answers Asked 6th Jun, 2018 Judith Mehrmann My goi is located in the nucleus and I cloned it into a YFP vector. The screening at the konfocal was successful and now I want to take a closer look at the nuclei. View How can I get attB PCR product. Question 4 answers Asked 21st May, 2018 Oscar Castaneda Helllo, I am workig with three elements Multisite Gateway Kit to clone a promoter, gene and terminator. Now I have good results for the promoters and terminator, even I could add attB regions for two of the four genes I have to clone.
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However two of the genes I could not get the attB PCR products. I have tried several times the PCR reaction testing different annealing T. Also I have tested several forward and reverse primers in which I have add more or less base pairs in the region in which the primers bind to the target gene but results always are the same, No attB PCR product appears. I really appreaciate if someone can give me advise View Any simple and quick yeast cell lysis protocol. Question 4 answers Asked 14th Dec, 2017 Muhammad Uddin Dear all, Can any one please help me with a simple yeast cell lysis protocol. I tried the following buffer but I am confused (0.1M NaOH, 0.05M EDTA, 2 Tween 20 and 2 B-mercapthoethanol). I used equal voulume of buffer with cell suspension, mix by vortexing and incubate at 90C for 10 min and centrifuge for 5 min at 12000 rpm to get the supernatants. Question 6 answers Asked 4th Dec, 2017 Allyson Cochran I'm attempting to probe for low molecular weight proteins (11-25 kDa) from whole-cell lysates using a Tris based RIPA lysis buffer with inhibitors. However, a dye 'blob' keeps forming in each lane at the gel front. The 'blobs' become more apparent as the separation proceeds and a small purple band also forms in front of them over the course of the run at constant 100V. The size marker runs fine, other than the smallest two marker bands run off the gel before the 'blob' reaches the bottom which I'm guessing means I've also lost the proteins I'm interested in. I FIGURED OUT THE PROBLEM!! The sample buffer we were using was the issue. Originally, we were using a 5x sample buffer with beta-ME and freezing 1ml aliquots. When a fresh batch was made, DTT, instead of beta-ME, was used; this is what was producing the dual-colored dye front. The solution that we've decided on is to start with aliquots of 5x NON-REDUCING sample buffer. Upon thawing a new aliquot, 25 beta-ME is added. This has completely resolved the issue. See a new image below.
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Ran the gel using a colleague's Tris-glycine separating (12) and stacking (4) buffers. My colleague and I also share the same 1x running buffer, other reagents and equipment. (I have my own power supply but have never had an issue with it on previous runs.) Made my own fresh Tris buffers and ran another gel with 6ul 5x sample buffer and 24ul of 1 Triton-X 0.25M 7.2pH Tris-glycine as a control against the RIPA again with NO PROTEIN. I've attached two images. I didn't have a picture of the gels, unfortunately, but the 'blobs' transferred to the membrane, which I have pointed out. I'm wondering: I just started using RIPA for lysis (same time the blobs appeared); also, could it be caused by a higher-than-normal molarity of Tris base in the running gel (0.75M rather than 0.5M)? The blob appears shortly after the samples enter the running gel. All DNA was removed prior to boiling. Neither of the gels had any yellow in them. Thanks again! View Total Protein Extraction protocol. Question 6 answers Asked 6th Oct, 2017 Samira Abdi im looking for a protocol which is specified for the extraction of whole protein (membrane, cytoplasm and nucleus). Can anyone help me w?th a rutin protocol on this?? View What should be the ideal extraction buffer composition for plant total protein isolation Question 6 answers Asked 9th Jan, 2017 Nazia Rifat I am using the extraction buffer for plant total protein isolation for MALDI.View LR Reaction problems? Question 5 answers Asked 6th Jan, 2017 Jenna Labelle I'm doing an LR reaction (gateway cloning) to move my gene from a pENTR (kan resistance) vector into the Dest8 (amp resistance) vector. I'm using TOPO10 cells, which are not resistant to the ccdb gene in the Dest8 vector, so I would think that only Dest8 vectors that switched out the ccdb gene for my gene would be able to grow. However, when I sequence for my gene, it's not there. Any insight into what may be going wrong. Thanks!
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View Any tips for critical parameters in the Gateway LR reaction. I have checked it 4 different ways: (1) Restriction mapping - was OK; (2) Ampicillin and Chloramphenicol resistance - was OK; (3) ccdB functionality - no colonies grew out from TOP10 or from Stbl3 cells when transfected, and (4) functionality of the Tet repressor-P2A-PURO cassette - transfected the plasmid into HEK and cells became PURO resistant. 3. After the LR reaction a lot of colonies grew out, and no colonies grew from the pLIX403 destination vector only. I tried another lentiviral vector (pINDUCER21) with the same result. Can anyone help me with some suggestions. Are there any critical parameters which I'm missing either during the LR reaction or during transfection or plasmid isolation. Thanks for the answers in advance View Related Publications Cloning vector pFA6a-HBH-kanMX6, complete sequence Data Feb 2006 P. Kaiser Christian Tagwerker On Jul 18, 2006 this sequence version replaced gi:89276250. View Got a technical question. Get high-quality answers from experts. Keep me logged in Log in or Continue with LinkedIn Continue with Google Welcome back. Keep me logged in Log in or Continue with LinkedIn Continue with Google No account. All rights reserved. Terms Privacy Copyright Imprint. Instead, you can choose a molecular cloning technique that will work well with a given set of resources, time, and experimental needs. Since its invention in the late 1990s, Gateway cloning technology has become very popular as a rapid and highly efficient way to move DNA sequences into multiple vector systems. With the appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety of expression systems. Keep reading to learn more about the Gateway cloning method and its advantages. I n vivo, t hese recombination reactions are facilitated by the recombination of attachment sites from the phage ( att P ) and the bacteria ( att B ).
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As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites ( att L -left- and att R -right-, Figure 1). Under certain conditions, the att L and att R sites can recombine, leading to the excision of the phage from the bacterial chromosome and the regeneration of att P and att B sites. Gateway technology relies on the two reactions described below: This reaction is catalyzed by the BP Clonase enzyme mix and generates the entry clone containing the DNA of interest flanked by att L sites. As a byproduct of the reaction, the ccd B gene is excised from the donor vector. This reaction is catalyzed by the LR Clonase enzyme mix. As a result, an expression clone with the DNA of interest flanked by att B sites is generated. As in the BP reaction, a DNA fragment containing the ccd B gene is excised from the destination vector. The entry clone and destination vector carry different antibiotic resistance markers (indicated here by plasmid color), allowing you to easily select for the expression clone. You will also need to use a E. coli strain sensitive to CcdB ( e.g. DH5?, TOP10, Mach1 ). The ccd B gene is present in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the gene of interest during the BP or LR reactions. Since the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined construct. Read our recent Plasmids 101 Post on CcdB for more information. If you choose this strategy, it’s important to include the proper protein expression elements (ribosome recognition sequences, start codon, stop codons, reading frame considerations, etc). This video demonstrates how to use the Snapgene program to design Gateway plasmids. TOPO cloning adds short end(s) to facilitate cloning into an att L -containing entry vector.
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This fragment is inserted in a multiple cloning site (MCS) of an att L -containing entry vector. This choice will depend on a number of factors, like your organism, desired expression level, and experimental purpose. The chosen att R destination vector will recombine with the at t L -entry clone to create the expression clone. Then, you can transform or transfect the cells that you want to use for your experiments and verify that your construct is functional. Addgene’s ready-made entry clones can be used with a large variety of plasmids. It is also possible to set up the BP and LR reactions in the same tube, speeding up the cloning of the att B -PCR products directly into destination vectors. The cloning process is simple - no restriction, ligation or gel purification steps are required! You can clone up to 4 DNA fragments, in a specific order and orientation, in one tube, into one Gateway vector to produce the desired expression clone. This is possible thanks to the Gateway vectors’ design. They have modified versions of the att B, P, L and R sites that recombine very specifically and directionally: att B1 sites react only with attP1 sites; attB2 only with attP2, attL1 only with attR1; attL2 only with attR2, and so on. Take a look at some of the Gateway Multisite plasmids available at Addgene, including the Frew Lab Multiple Lentiviral Expression Systems (MuLE) Kit, the MultiSite Gateway cloning kit, and MultiSite Gateway plasmids. These can be used to express genes in a variety of model organisms. Use the links below to find Gateway plasmids for your organisms of interest: Curr Protoc Protein Sci. 2003 Feb;Chapter 5:Unit 5.17. PubMed PMID: 18429245. We archive and distribute high quality plasmids from your colleagues. Version (.PDF) If you want to optimizeHere we use Electroposation requires low concentrations All our Binary-vectors are spectinomycin resistant. If you are You can obtain To check for Please do not reproduce this. Do not unzip the tutorial.
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Gateway Cloning Tutorial Note: To complete the tutorial with the referenced data in Geneious Prime please download and install the tutorial above. Introduce your expression clone into the host system of choice for expression of your gene of interest. In this tutorial we will learn how to: Design Gateway-compatible oligonucleotide primers to amplify a sequence of interest Simulate PCR to create a Gateway-compatible PCR product Simulate recombination of PCR product with a donor vector via a “BP” reaction to create an Entry vector Simulate recombination of an Entry vector with a Destination vector via an “LR” reaction to create an Expression vector Simulate multipart Gateway cloning Tutorial Structure The first three exercises in this tutorial cover the steps required for simulation of single insert Gateway cloning. The fourth and fifth exercises cover simulation of multisite Gateway cloning. The final section describes how to prepare vector sequences for use with the Gateway cloning tool. The two nucleotides cannot be AA, AG, or GA, as these additions will create a termination codon If fusing your sequence in-frame with an C-terminal tag, the reverse primer must include one additional nucleotide to be in-frame with the attB2 region. In future versions of Geneious Prime the “spacer” nucleotides will be added as separate annotations. The primer design tool will not add translation initiation motif sequences. Users will need to add these motifs manually as extensions during the primer design process. The primer design tool will not add a stop codon to a reverse primer. If you require a stop codon you should incorporate a template-derived stop codon (if present) or manually add a stop codon as an extension during the primer design process.
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Exercise 1: Designing primers for Gateway cloning Exercise 2: Simulating an “BP” Entry clone reaction Exercise 3: Simulating a “LR” Destination clone reaction MultiSite Overview: Overview of Gateway multisite cloning Exercise 4: MultiSite Part 1: Primer design and PCR Exercise 5: MultiSite Part 2: Creation of Entry and Destination vectors Gateway Vectors: Preparing Gateway vector sequences Exercise 1: Designing Primers for Gateway Cloning In this exercise we will design oligonucleotide primers to amplify the mature xynB CDS. The forward and reverse primers will be designed to incorporate attB1 and attB2 sites respectively, to allow clonase-mediated integration of the PCR product into a Gateway entry vector. This will ensure optimal transcription and expression of the XynB gene product. Click OK, and you will see that the extension will comprise 4 G nucleotides and an attB1 site. Click to run the Primer design tool and two new annotations, 139 F and 1149 R will be added to the sequence. Hit Save (Command or CTRL-S) to save the new annotations. Hover over each primer sequence and you will see a yellow tool tip containing information about the primer. 7. The next step is to generate a PCR product sequence. Select the DTU76545 file, click on the Primers button and choose Extract PCR Product. The tool will detect the new forward and reverse primer annotations on the sequence and select them. In the dialog that opens, confirm the Forward and Reverse primers are correctly selected, then hit OK. A new DTU76545 PCR Product sequence will be created. Select it and you should see it has inward facing attB1 and attB2 sites positioned and oriented correctly for a BP clonase reaction. We will use this PCR product in the next Exercise. If you plan to routinely design Gateway Primers then you can save your ideal primer design settings by clicking on the cog icon in the bottom left corner of the Design New Primers window and choose to Save current settings.
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You can load these settings next time by clicking on the cog icon and choosing Load Profile. You can use these extracted sequences for ordering from your oligo synthesis service provider. Exercise 2: Simulating a “BP” Entry clone reaction In this exercise we will simulate a BP clonase reaction between our new PCR product and a donor vector with appropriate attP1 and attP2 sites. Select the pDONR221 sequence to view it. You will see this vector has attP1 and attP2 sites flanking a chloramphenicol ( cmR ) resistance gene and a ccdB toxin gene. This region will be replaced by recombination with the PCR product to create an entry vector. The Gateway cloning tool will identify the att sites present on both the vector and the PCR product insert, confirm they are appropriately oriented, and inform that a BP reaction will be performed. This new 3558 bp sequence represents the expected entry plasmid generated by the clonase reaction. You will see that the clonase reaction has created flanking attL1 and attL2 sites suitable for use in a clonase-mediated LR-reaction. The pDONR vectors manual recommends that individual E. coli colonies containing your clonase-derived constructs are checked by PCR using the M13-F (-20) and M13-R primers. Binding sites for these primers are present and annotated onto the pDONR221 vector. We will now use the Extract PCR Product tool to simulate this PCR reaction so that we can determine the size and sequence of the predicted product. Set the Forward primer to M13-F (-20) and the Reverse primer to M13-R (-26). This will create a 1303 bp PCR product sequence. Select the pDEST17 sequence to view it. You will see this vector has attR1 and attR2 sites flanking a chloramphenicol (cmR) resistance gene and a ccdB toxin gene. The Gateway cloning tool will identify the att sites present on the entry vector and Destination vector and confirm an LR reaction can be performed. In the test tube an LR reaction creates two new plasmid species.
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The Gateway tool will output both plasmids if you wish. The tool will ask if you want to “keep both products of the reaction”. In this exercise leave this option unchecked so only the final Expression vector will be created. The tool will confirm sites in each vector are appropriately oriented, and inform that a LR reaction will be performed. The new file name, although descriptive is a bit long and cumbersome. Select the file in the document table, click on the file name to edit it and rename the file to pDEST17:xynB mature. As a last step we will check that our insert is in-frame with the vector start codon and HIS-tag. Click on the xynB mature CDS annotation to select it, then drag the left hand boundary of the annotation to extend it to line up with the first “A” of the start codon. Note that changing annotations on a sequence with actively linked parents will break the linkage. When you adjust the xynB mature CDS annotation boundary you will get a warning about Actively linked parents. Performing the BP and LR reactions in a single step To simplify your Gateway reactions it is possible to use the Gateway cloning tool to perform the BP and LR reactions in a single operation. The Gateway cloning tool will confirm the selected sequences can be “reacted” and will ouput the same final 5713 bp expression plasmid. Overview: Simulating Gateway multisite cloning Thermo Fisher Scientific provide a number of specialised pDONR vectors that allow multiple “parts” (2, 3 or 4) to be recombined, in a specific order and orientation, with a destination vector. The Geneious Prime Gateway Cloning tool allows simulation of multisite Gateway reactions. The steps required for multisite cloning parallel those performed in Exercises 1, 2 and 3: Design primers for PCR amplification and addition of unique attB sites to each part. Create individual entry clones for each part using specific multisite pDONR vectors.

Recombine the 3 entry clones with a destination vector using the Gateway cloning tool. The Gateway clonase recognition sites are directional and must be orientated appropriately in order for multisite recombination to proceed correctly. The following table is provided to assist when using the Geneious primer design tool to ensure you select the correct att sites when adding to primers for amplification of multisite Gateway parts. Select this folder to see the example files required for this exercise in the Document Table. As part of the work described by Peterson and Stowers (2011), a Gateway multisite reaction was used to place the yeast GAL4 CDS between two regulatory elements derived from the D. melanogaster trpA1 gene. The final Gateway construct was then integrated into into the D. melanogaster genome to demonstrate that the trpA1 regulatory elements upregulated GAL4 expression, and consequently UAS-GFP (green fluorescent protein) expression, in larval sensory neurons. In this exercise we will simulate the work of Peterson and Stower and use the Geneious Gateway tool to perform a Gateway multisite reaction to combine separate upstream and downstream D. melanogaster trpA1 regulatory elements (trpA1 UP and trpA1 Down) with the yeast GAL4 transcriptional activator CDS and with the D. melanogaster -specific destination vector pDESTHaw. The required part order (with att sites used) is depicted below: In this exercise we will: Design primers for PCR-mediated addition of unique attB sites to each of the trpA1 UPstream, GAL4, and trpA1 DOWNstream parts. Recombine the 3 entry clones with the destination vector pDESTHaw, using the Gateway cloning tool, to create a sequence suitable for PhiC31 integrase (attB)-mediated integration into the genomes of D. melanogaster embryos. As per the table in the Multipart Overview we need to use PCR to add flanking attB1 and attB4 sites to the first trpA1 UP sequence.

To successfully design PCR primers to amplify this sequence using the Design new Primers tool we will need to adjust the default Primer Design settings. Select the trpA1 UP sequence, click the Primers button in the Toolbar and choose Design new Primers. Use the settings shown below. This PCR product will be used in the next section for recombination with the entry vector pDONR221 P1-P4. Step 2: Primer design for GAL4 CDS sequence The GAL4 CDS will be the second part of the 3 part multisite reaction. As per the table in the Multipart Overview we need to add flanking attb4r and attb3r sites to the GAL4 sequence. Select the GAL4 sequence file, and click the Primers button in the Toolbar and choose Design new Primers. Perform the same steps as in Step 1, this time adding a sense attB4r site to the forward primer and an antisense attB3r to the reverse primer. This PCR product will be used in the next section for recombination with the entry vector pDONR221 P4r-P3r. As per the table in the Multipart Overview we need to add flanking attB3 and attB2 sites to the trpA1 DOWN sequence. Select the trpA1 DOWN sequence file, and click the Primers button in the Toolbar and choose Design new Primers. Perform the same steps as those in step 1, this time adding a Sense attB3 site to the forward primer and an antisense attB2 to the reverse primer. This PCR product will be used in the next section for recombination with the entry vector pDONR221 P3-P2. You should now have three PCR products ready for integration into donor vectors. Exercise 5: Multisite Part 2: Creation of Entry and Destination vectors Part 2: Creation of Entry vectors We will now combine our new PCR products with the appropriate donor vectors using the Gateway Cloning tool. We now have 3 pDONR entry vectors that can be recombined with an appropriate destination vector in a multisite reaction.

Part 3: Creation of the final destination vector To create the final destination vector, select pDESTHaw vector and the three new entry clone sequences. Performing the multisite BP and LR reactions in a single step To simplify your Gateway multisite reactions it is possible to use the Gateway cloning tool to perform all BP and LR reactions in a single operation. The Gateway cloning tool will confirm the selected sequences can be “reacted” and create the same 15,894 bp pDESTHaw-based expression plasmid. Gateway Vectors: Preparing Gateway vector sequences The Thermo Fisher Scientific website provides the sequences for their Gateway vectors. However they are not available in a useful annotated form. If you obtain and import an unannotated Gateway vector sequence then you can use the Geneious Annotate from: tool to identify and annotate various features onto the sequence. For more information our tutorial on Transferring annotations which can be downloaded from. The Geneious Gateway cloning tool requires that Gateway vectors and inserts are annotated with special att annotations. For example, an attR1 motif should be annotated with an annotation of Type: AttR1 and Name: attR1. If you receive an annotated Gateway sequence that appears to have att annotations, but it is not accepted by the Gateway cloning tool then double check each att annotation and ensure the correct Type is set. Privacy Policy Legal Download Case Study Notice: JavaScript is required for this content. X Download NGS eBook Notice: JavaScript is required for this content. X Download White Paper Notice: JavaScript is required for this content. X Notice: JavaScript is required for this content. X. Gateway Cloning Technique allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. Using Gateway, one can clone subclone DNA segments for functional analysis.

The system requires the initial insertion of a DNA fragment into a plasmid with two flanking recombination sequences called “att L 1” and “att L 2”, to develop a “Gateway Entry clone” (special Invitrogen nomenclature).The availability of these gene cassettes in a standard Gateway cloning plasmid helps researchers quickly transfer these cassettes into plasmids that facilitate the analysis of gene function. Gateway cloning does take more time for initial set-up, and is more expensive than traditional restriction enzyme and ligase-based cloning methods, but it saves time, and offers simpler and highly efficient cloning for down-stream applications.Entry clones are often made in two steps:The enzyme mix catalyzes the recombination and insertion of the att-B-sequence-containing PCR product into the att P recombination sites in the Gateway Donor vector.Thousands of Gateway Destination plasmids have been made and are freely shared amongst researchers across the world. Gateway Destination vectors are similar to classical expression vectors containing multiple cloning sites, before the insertion of a gene of interest, using restriction enzyme digestion and ligation. Gateway Destination vectors are commercially available from Invitrogen, EMD ( Novagen ) and Covalys.By using this site, you agree to the Terms of Use and Privacy Policy. 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.If you run low, you can always just miniprep again. 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.Where more than one catalog number is listed, this reflects the different sizes available.Note: unlike BP Clonase, which had a separate buffer, BP Clonase II includes the buffer in the enzyme mix.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.)Note: do NOT use One Shot TOP10F' cells; these will not show a difference in clear and opaque colonies (see below).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.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.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.This list of att site sequences may be useful.For each, PCR was performed in a 50 ul reaction.The appropriate band is excised and DNA purification performed using the Qiagen Qiaquick Gel Extraction Kit (for DNA fragments 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.