...library, or by RT-PCR. Isolate the PCR product Isolate your PCR product from the rest of the PCR reaction using...-3’. Preparing the PCR product for cloning Run the PCR reaction Run PCR to amplify your insert DNA. It...such as the QIAquick PCR Purification Kit. The PCR product is now ready for restriction digestion. As such...and to also be compatible with a standard PCR reaction (see PCR Video). Therefore, our Forward Primer will... to be amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying from a...design and the PCR process itself are very similar to those outlined in our restriction cloning post with...Your Gene of Interest (YGOI) for ligation into a recipient plasmid. Designing primers for PCR based cloning...

...easy to amplify by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not ...matter if the DNA is PCR amplified, restriction digested or even a synthesized gene. PCR is the primary method...included in a single PCR, for example for subcloning genes from different templates. All PCRs are run as an ...errors during PCR: we recommend Phusion or Q5. Speed up your life Rather than making up your PCR mix every...side of the undesired portion, designed to amplify the rest of the vector sequence. Encode homology in...another simply amplify the vector and insert with separate primer pairs in the same PCR. Add homologous...the necessary primers to the same PCR mix. Pairs of primers will amplify the whole vector sequence between...

... of PCR   Type of PCR Goal of the PCR method Uses of PCR method End point PCR or just PCR Amplify... our PCR protocol page, protocol video and reference on how to design PCR primers.  Types of PCR Since...applications. These PCR methods all use the same basic PCR set up and steps but differ in how the PCR products are...analyzed.   End point PCR End point PCR, as the name implies, analyzes the end product of PCR temperature cycling...post “Droplet Digital PCR for AAV Quantification.”   Multiplex PCR Multiplex PCR, as the name implies,...targets can be amplified in a single PCR experiment using multiple primers all in one PCR reaction. This...categories of multiplex PCR:  Single template PCR reaction - one template is amplified using several forward...

...insert and vector are first PCR amplified. The forward primer used to amplify the vector is located in the...2011 to the right. After PCR, you should confirm that the products were amplified by running a small aliquot...ve confirmed that the PCR was successful, mix together the vector and insert PCR products with DpnI, and...Addgene Blog Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background...Cloning Choose Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - ...cloning, one step cloning, and overlap extension PCR. Although each type of cloning has its advantages... techniques: unwanted mutations due to excessive PCR cycles or low fidelity Taq DNA polymerase, the construction...

...primers (with the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent...plasmids. PCR primers (green) amplify the plasmid template (blue), and introduce a restriction site “A*...efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified fairly easily...: After the PCR reaction, no ligation is required since the E. coli you transform your PCR products into...shown are the “B” restriction sites, which flank the targeted region. Upon PCR amplification, the template...results (left) and validation PCR followed by Restriction Fragment Length Polymorphism (RFLP) analysis (right... successfully anneal to the plasmid of interest during the PCR reaction. Ideally, your primers should ...

...colony PCR.   Designing colony PCR primers The first and perhaps most important step to colony PCR is designing...   PCR set-up Setting up colony PCR reactions is nearly identical to preparing a standard PCR reaction...colony PCR are: 1) design primers to detect the presence of your insert; 2) set up a standard PCR reaction... in order to serve as PCR template. Dealing with this and a few other colony PCR tips are highlighted ... up PCR reactions: The remaining bacteria-water suspension will serve as the template for your PCR reaction...sample to the PCR reaction. The bacteria will be lysed during the initial heating step of the PCR reaction....best controls for a colony PCR are the same ones used to verify if the colony PCR primers work in the first...

...insert Sequence the regions that have been amplified by PCR, putting special attention in the areas of...are having difficulty amplifying your target sequence, or if you want to amplify sequences from a large...Addgene. Cloning can be quite an arduous process. The PCR could fail to produce a product, the transformation... best to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least 24 bp with the...primer3) or provided by the major companies selling PCR reagents. Nowadays commercial polymerases are very...recheck your primer design or optimize your primers or PCR conditions. Purifying DNA fragments Once you’ve ... good habit to purify your digested fragments or PCR products from a gel. You will avoid contamination...

...together multiple PCR products using sequence homology is also common. Overlap extension PCR is used to splice...with the desired mutation, then use those to PCR amplify the template plasmid. The parental, nonmutated...pitfall of REPLACR-mutagenesis is that you must PCR amplify the whole plasmid, which could introduce mutations...Recombineering of Ends of Linearized Plasmids After PCR). Current site directed mutagenesis methods Current... DNA-specific enzyme DpnI, and the nonmethylated PCR products are then transformed into E. coli to undergo...splice together two PCR amplicons based on sequence homology, and the subsequent product is then digested... DpnI to digest away the template plasmid, these PCR products are directly transformed into bacteria expressing...

...need to amplify a library for your own experiments. Library amplification 101 Library amplification seems...samples using both pre- and post-amplification DNA and generates PCR sequencing products from both. These...exciting and versatile offerings! We recently re-amplified our distribution stock of the Brunello Human gRNA...taken to ensure that all of these plasmids are amplified and none are lost. Each Addgene library has its...are differently sized, smaller plasmids may be amplified preferentially, skewing the composition of the...primers to create 200-300 bp sequencing products using PCR. A diagram of the product we created for the Brunello...library is shown in Figure 2. You can use a small PCR product because the plasmids in the library are identical...

...subject to reverse transcription. This is then amplified by PCR using Taq Polymerase and then transcribed ...Highlights: Reverse-transcribed RNA samples are amplified by PCR instead of RPA. Unlike RPA, this technique... the typical PCR machines, the authors use affordable, field-ready, Bluetooth-enabled PCR machines that...SARS-CoV-2 nucleic acids, the target sequence is amplified by PCR/RPA using biotinylated primers. These are ...and Cas13 is then used to detect the amplified region of interest. Cas13 uses a complementary crRNA sequence...case, regions of gene S and Orf1ab gene are amplified. RPA reactions also contain reverse transcriptase to...resulting in fluorescence. Cas12a binds all the amplified target regions and results in the continuous cleavage...

...BRAF gene edit, simply PCR-amplify the BRAF locus from the edited cells (the PCR product should contain...edited BRAF sequence), and PCR-amplify the BRAF locus from control cells (the PCR product should contain ...t in the middle of the PCR product. If the mismatch is in the middle of the PCR product, you would expect... monoclonal cell lines by limiting dilution. The rest of the cells were frozen down. Figure 1: ...primers flanking my edits in genomic DNA and doing PCR on genomic DNA. The surveyor assay itself was relatively...contain the wild-type BRAF sequence). Then, mix the PCR products and use temperature to denature and renature...genome browser and Primer3 to design primers that amplified a 500-600 bp product. The primers I designed (...

...performed to amplify the target using PCR. Instead of tracking the reaction progress as in real time PCR, each...that will be elaborated below. Digital PCR Digital PCR (dPCR) involves splitting up a sample into thousands...we’ll introduce digital PCR as an emerging validation technology. Digital PCR has several advantages over...sample. Digital PCR assays for mutation screening Now that we have introduced how digital PCR works, let’s... 200 and 500 ng of purified PCR product. Low detection limits: Digital PCR assays can accurately quantify... (Figure 3). If you are interested in harnessing the power of digital PCR screening to validate your...Hauber, I. & Fehse, B. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases...

...Gibson assembly. In this case the vector was PCR amplified to contain regions of homology with the bacterial...neighboring fragment. Then you would amplify the fragments and vector by PCR, verify that you have bands of ...CRISPR cutting was used to circumvent the need to PCR amplify the fragments of the chromosome, which was technically... neighboring fragments can be created via PCR amplification with primers that contain the appropriate ...have tools that help you design primers for PCR amplification of fragments to incorporate such regions of... which could make the PCR step difficult--OR there is no convenient restriction site for linearization...2015). In this case, rather than using a restriction enzyme or PCR to make linearized vector, the Cas9 enzyme...

...to perform PCR in step 6.3 . Assemble a 20 μl PCR with the following components: 10 μl 2x PCR mix, 0.5 ...each well) to a 96-well PCR plate using a multichannel pipette. Centrifuge PCR plate at 400 x g for 5 ... in prostate tumors by quantitative real-time PCR (qPCR). Gordanpour A, Nam RK, Sugar L, Bacopulos...biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility ... genomic DNA (gDNA) for the intended deletion by PCR (see steps 6.1 and 7.1 ). The bulk cells have sufficiently...sgRNA-B (see step 5 ). The purpose of the following PCR is to validate primers and verify the presence of...Use the primers designed in step 2 above. Conduct PCR for “non-deletion band” and “deletion band” in separate...

...start the SLIC cloning process, a gene of interest is PCR amplified to add 5′ and 3′ homology regions from...by annealing two separate PCR products (middle branch); or by incomplete PCR, which skips the final extension...single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product... assembly with SLIC. Multiple fragments are PCR amplified to add 5′ and 3′ homology regions to the adjacent...tube. Alternatively, SLIC can also work directly on PCR products, with 5′ overhangs generated either by annealing...annealing a mixture of products or from incomplete PCR — although higher DNA concentrations are needed for...flexibility allows it to be used with different types of PCR-generated inserts. By harnessing the power of DNA...

... process, or visit our restriction analysis blog post. Colony PCR Colony PCR can detect the presence...negative controls for your PCR.  Find the details and learn more about colony PCR in this blog post.   ... but amplify a potential insert. If you’re using insert-specific primers, you should expect a PCR product...your plasmid. In this case, you’d be using colony PCR to detect your insert, either by using primers that...product if the plasmid contains the insert and no PCR product if the plasmid does not contain the insert... Figure 2: The steps of colony PCR.   Sanger sequencing Sanger sequencing determines the precise...no insert in the plasmid, you won’t be able to amplify anything. Like any experiment, you’ll want the ...

...mixed cell population. The region of interest is PCR amplified, PCR products are denatured-renatured, treated...that introduce a novel restriction site. The region of interest is PCR amplified, digested with the appropriate...region of interest can be PCR amplified using primers that (A) flank the region of interest (deletions... The PCR product is then run on an agarose gel to determine whether the edit was successful. PCR amplification...appropriate restriction enzyme and run on an agarose gel to identify DNA fragments. PCR amplification and gel...an agarose gel to identify DNA fragments. PCR and restriction digest (for HDR repaired DSBs): For small...exact sequence of targeted alleles. Involves PCR amplification of targeted region from DNA, subcloning into...

...to focus on PCR-based applications (qPCR and ddPCR.) qPCR Quantitative/real-time PCR (qPCR) has been used... courtesy of UPODMG 1516 irivbrro ddPCR  Droplet digital PCR (ddPCR) works by using oil droplets to partition...used for copy number assays for years. qPCR works like regular PCR, just with signaling molecules added ...the reference sequence and the gene of interest to be amplified in the same reaction. Taqman’s increased...partition a single sample into thousands of PCR reactions using Taqman chemistry (see image, left, of ...Complexity of the gene of interest Some gene sequences are harder to amplify and quantify than others.... Resources on the Addgene blog   Droplet Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview...

...and then we performed colony PCR on the register region and sent the PCR product for sequencing.  A less...register enables the design of primer pairs that PCR amplify in some states but not others. Because samples...one state, regular PCR may not be very informative – each primer pair will amplify even if its target ...Instead, one may use qPCR to determine the fraction of registers that are amplified by a particular primer...three-input GRSMs, we recommend using overlap extension PCR and Gibson assembly to piece together the registers... individual parts and/or strings of parts may be PCR-ed out of their current registers and used to build... expensive, then the qPCR method should be considered as an alternative. The qPCR method can be performed...

...insert by PCR, restriction digest, or sequencing. Pro tips Do not add 5’ phosphates to your PCR primers...ends) and amplify your sequence of interest with Taq polymerase using your favorite PCR protocol.  2. ... after the last cycle of PCR to make sure that the "A" gets added to all PCR products. Keep in mind ...method that does not use restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy...overhang on the blue PCR product insert comes from using Taq polymerase for the amplification step since Taq...1. Create Your PCR Product: Design standard primers (no need to add unique restriction sites on the ends...leaves a single deoxyadenosine (A) at the 3' ends of PCR products. The complimentary “T” in the pair comes...

..., performing PCR to generate the mutation and linearizing your vector (preferably by PCR) for DNA assembly...Regions of homology can be introduced to fragments via PCR using primers that contain the regions of homology...fragments are added, deleted, or reordered and PCR enzymes are adjusted. Appropriate fragment and primer design...scarless DNA – perfect for transforma­­­tion or as a PCR template. Some of the applications of the NEBuilder...selectable marker, and target gene, for example, are amplified with overlapping regions of homology and inserted... Based Cloning Methods Screen Plasmids by Colony PCR Addtional Resources on Addgene.org Browse Our CRISPR...standardized DNA constructs, but its use of Type IIS restriction enzymes results in scarless assembly. Finally...

...simply PCR-amplify your vector and your insert, DpnI digest away the templates, and transform the PCR products...coli then fuse together the PCR products via overhangs left over from the PCR process (the precise mechanism...generating the appropriate inserts by PCR, there is no need for restriction digests, gel purifications, or ligations... In this technique, you first perform a round of PCR to generate DNA fragments with 15-40 bp of homology...particularly tight? Most cloning processes will require a PCR reaction or two to generate templates for the cloning...of all the options discussed as it only requires PCR and the inexpensive DpnI enzyme. Otherwise you let...to cloning different types of DNA, such as cDNA, PCR fragments, or genomic DNA. Also remember that commercially...

...used as a template for PCR amplification with a degenerate primer set to amplify IgG variable light (VL... then verified by both diagnostic restriction digest and colony PCR.   Fig. 1: (A) Schematic of... and variable heavy (VH) chain sequences. Fusion PCR was performed to create an amplicon that  was cloned... for some constructs more than 90% of the colony PCR validated clones unexpectedly failed to produce functional...chain sequences. By performing a BciVI digest of the PCR products prior to cloning, which in most cases left...activated B cell that produces the antibody of interest with a myeloma cell. This RNA was then used to... found that the recognition sequence for the restriction enzyme BciVI was present in the aberrant light...

...Error-prone PCR: This method amplifies the wild-type template with a “sloppy” version of PCR that results...mutagenesis (SDM) PCR, but with a twist since it relies on the sequential nicking, degradation, and PCR-generation...a universal primer is used for this round of PCR. The PCR product is digested with DpnI to remove residual...that tile across a region of interest, generating multiple mutants in one PCR reaction. Below is a step-...of up to 2% per nucleotide position. The “sloppy” PCR reaction is created by making some or all of the ...increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2...ssDNA as template for a site-directed mutagenesis PCR that uses mutagenic primers to introduce all desired...

...perform PCR against components of the viral vector using the harvested viral solution as a PCR template...virus-based experiment at one point or another. After amplifying a viral vector, collecting virus, and transducing...replication. To avoid genomic rearrangements, try amplifying the viral vector with bacteria designed to minimize...integrity of your virus, perform a diagnostic restriction enzyme digest and compare it to some reliable... use the virus immediately upon harvesting. Interestingly, freeze-thawing host cells immediately prior...24003179. PubMed Central PMCID: PMC3760253. 2. Rohwer, Forest. "Global phage diversity." Cell 113.2 (2003): 141...

...TIDE First, use PCR to amplify the target region of interest. Then Sanger sequence the PCR product of both...screening by PCR may be the way to go. Design a PCR to flank your desired edit site and run the PCR product... can screen using PCR and a restriction enzyme digest.   Fig. 3: Restriction enzyme screening ...your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs of sequence... you’re in luck! You don’t need to sequence your PCR to visualize an edit. Most dual guide systems generate...enough that it can be visualized by running your PCR on a gel. It’s still paramount that you sequence ...or deleted accordingly. Pro tip: make sure your PCR amplicon isn’t too large so that you can easily visualize...

...definition) in a specific field.  For example, while “PCR test” has recently transitioned from jargon to non-jargon...jargon in most other uses. Many laypeople would use “PCR test” to mean “a test that uses a biological sample...sequence that uses trprimers and enzymes that will amplify a sequence only if present.” You can see how you...of knowledge or interest in STEM subjects; we generally refer to them as the “interested public.” Subscribers...to Scientific America would be considered the interested public and it would be safe to assume that they...age range, familiarity with the subject, shared interests, socioeconomic and/or racial/ethnic background...example adults 45-55 yrs old who breed or are interested in breeding dogs who also enjoy listening to ...

...the cellulose directly into a PCR buffer, where DNA or RNA can be amplified. Compared to a commercial magnetic...purification Starting material: PCR product or agarose gel slices Product: purified PCR product or DNA fragments...method for regenerating silica columns that come with PCR product purification or DNA gel extraction kits. ...Rapid Regeneration and Reuse of Silica Columns from PCR Purification and Gel Extraction Kits. Scientific ...was free of agarose and could be digested with restriction enzymes.  Cellulose nucleic acid purification... still be eluted from the column, as detected by qPCR, but this did not reduce cloning efficiencies.  ...

... cDNA library, or by RT-PCR. Isolate your PCR product from the rest of the PCR reaction using a kit, such...Experimental Procedure Run PCR and Ppurify the PCR Product Run PCR to amplify your insert DNA. It is important... Protocols Plasmid Cloning by PCR Plasmid Cloning by PCR You may also like... Restriction Digest...window) QIAquick PCR Purification Kit . The PCR product is now ready for restriction digestion. Digest... your PCR product is the anticipated size and that your band is strong (indicating that the PCR reaction... to be amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying from a...Your Gene of Interest (YGOI) for ligation into a recipient plasmid. Designing Primers for PCR Based Cloning...

...Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus to verify correct GFP-... required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins...microhomologies are added to the GFP-Puro cassette via PCR, and this construct is inserted into the PITCh vector...target near the last coding exon of your gene of interest. For ideal use, this gRNA should be cloned into...endogenous promoter and the 3’ UTR of the locus of interest, since the GFP-Puro will be inserted just upstream...nucleases and their applications. It will be interesting to see what new editing possibilities MMEJ can...

... the sequence of interest are annealed to target DNA (genomic DNA or a PCR amplified fragment) and treated..., Francis. "The ligase chain reaction in a PCR world." PCR Methods Appl 1.1 (1991): 5-16. PubMed PMID:...chain reaction (LCR)-overview and applications." PCR Methods Appl 3.4 (1994): S51-64. PubMed PMID: 8173509...bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis.... 18. Hommatsu, Manami, et al. "Development of a PCR/LDR/flow-through hybridization assay using a capillary...Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources on Addgene.org Basic Molecular Biology...joining for recombinant plasmid construction (restriction cloning), adaptor ligation for next generation...

...base pair sequence surrounding your gRNA. Once amplified, you can insert this region into a pCAG-EGXXFP...primer sets to use to genotype your pups. A simple PCR with these primers will let you know if there are...are any major deletions, and sequencing of the PCR products will give specific details on what exactly ...alleles are present in these mice, you can clone the PCR products into a pBluescript cloning vector and sequence... delete the entire gene? Perhaps you are more interested in the c-terminus and would like to target that...

...better for PCR and surveyor. I used DNA polymerase Takara Ex Taq ™ DNA Polymerase for my genomic PCR, but couldn't...conditions with 5% DMSO or without 5% DMSO. My genomic PCR didn’t work, do you have any suggestions? The Zhang...the protocol, then uses around 2-4ul in each 50ul PCR reaction. The gDNA extracted in this way is usually...groups have successfully used Herc II or Kapa Hifi in PCR. Maybe this Takara enzyme is not very robust in this... couldn't amplify the EMX1 gene using same primer you used in the Science paper ( Cong et al. , Science...template, you could then: Confirm your HR by doing Restriction Fragment Length Polymorphism (RFLP) ( see Figure...

...Reaction Plasmid Cloning by PCR Agarose Gel Electrophoresis Primer Design for PCR Oligonucleotide primers ...Tips on designing primers for PCR Protocols...primers are necessary when running a PCR reaction. One needs to design primers that are complementary to the...On average, the DNA fragment that needs to be amplified should be within 1-10 kB in size. The structure...protocol video below to learn how to design primers for PCR. Reference Page Top Index...important as well. Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other...other hand, a long primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not...

...inserts can be made by PCR using primers that amplify the DNA sequence of interest and flank it with 50 ...modification. Colony PCR can be used to screen for positive clones in most cases, and restriction enzyme digest...recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates can be generated...ordering synthetic oligonucleotides or denaturing short PCR products. Either way, the substrate should be ~70...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an... of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from ...

...edited and unedited (control) populations is PCR-amplified and Sanger sequenced. ICE compares these sequence...sample names and guide sequences, and ICE will do the rest. There are no parameters that need optimizing and...This score is a useful measure for those who are interested in understanding how many of the contributing...

...from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase...transcription to convert amplified DNA to RNA for subsequent detection by Cas13. This amplification step in combination...sensitivity, the DNA is first amplified through isothermal amplification by RPA. When Cas12a-cRNA base...nuclease, can be used in conjunction with Cas13 to amplify the signal of a single target. Csm6 can cleave ... the Cas13 reporter), they could significantly amplify the detection signal for a single target. SHERLOCKv2...laboratories. In the case of the COVID-19 pandemic, qPCR can be used to diagnose the presence of SARS-CoV...presence and quantity of DNA, RNA or a mutation of interest. Together SHERLOCK and DETECTR demonstrate the...

...Reaction (PCR) Polymerase Chain Reaction (PCR) You may also like... Plasmid Cloning by PCR Restriction Digest... how to perform Polymerase Chain Reaction (PCR). Basic PCR Program Initial Denaturation for 2 minutes ...strands. Materials List Reagents for each 50µL PCR: Thin-walled PCR tube Ice Bucket 2 μL Template DNA (10 ng...Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer Design and PCR Design Primers. See our...volume of master mix to add to each PCR tube. Secure the tops to the PCR tubes, gently tap each tube to bring...cloning by PCR to learn how to design primers for cloning purposes. What do I do if my PCR isn't working...Information A standard Polymerase Chain Reaction (PCR) is an in vitro method that allows a single, short...

...and clump together ~3 days post infection. The amplified virus can be harvested once roughly 50% of the...the purified virus should be ~1.3 Quantitative PCR: the number of viral DNA packaged in virions is determined...for amplification. The final purified plasmid stock should be analyzed by endonuclease restriction digest...transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of AdVs is what makes... in trans. The Gutless AdV have generated high interest for gene therapy due their increased cloning capacity...AdV can be found in reference 3. Production, amplification, and quality control of 2nd generation AdV   ...plasmids: a shuttle vector (in which the transgene of interest is cloned) and pAdEasy™ which contains the adenoviral...

...titrated following purification using qPCR or digital droplet PCR. Insect cells Insect cells are a less... P0), infecting new Sf9 cells, and getting an amplified P2 virus. This P2 virus can be produced in a greater...plasmids — a transfer plasmid containing your gene of interest plus two packaging plasmids (three packaging plasmids...