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What are flagged primers?

What are flagged primers?


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I'm interested in amplifying a sequence for further use with Gibson Assembly. I want to create overhang regions in my DNA fragment so there would be complementarity to the plasmid I'm trying to insert it in.

I was informed by a colleague to work with "flagged primers" though I can't find any literature online. Can anyone explain this concept to me?


PCR Primers for introducing tag - (Sep/22/2008 )

Hi, I have recently cloned my wildtype gene in a pUAST vector. I want to introduce HA tags right before the gene and right after the gene(as two separate constructs). I also want o make sure I have a Kozak sequence and an A at the -3 before the tag. How can I do this by PCR. Can anyone suggest?

It's really easy! The HA tag is about 27-30bp, isn't it? Just order longer oligos then.

5'-(3random bp)-(Restriction enzyme site)-(HA tag)-(start of your gene (20-30bp))

3'-(end of gene)-(HAtag)-(RE site)-(3 random bp)-

After the PCR cut the product with restriction enzymes and paste in the place of your unmodified gene.

Usually the region, complementary to the gene is around 23-26bp. You can add your Kozak in front of the HA tag. Take care of the START and STOP codons if you want the tags to be translated.

I do it like this all the time. I have added tags as long as 60bp with primers. In such a case I just ordered a 85bp oligo (HPLC purified) and no problem. I usually use Pfu for such PCR.

Thanks Ramses. Will the PCR work if my plasmid is

10kb? IS there a good Polymerase that will not introduce random mutations?

It's really easy! The HA tag is about 27-30bp, isn't it? Just order longer oligos then.

5'-(3random bp)-(Restriction enzyme site)-(HA tag)-(start of your gene (20-30bp))

3'-(end of gene)-(HAtag)-(RE site)-(3 random bp)-

After the PCR cut the product with restriction enzymes and paste in the place of your unmodified gene.

Usually the region, complementary to the gene is around 23-26bp. You can add your Kozak in front of the HA tag. Take care of the START and STOP codons if you want the tags to be translated.

I do it like this all the time. I have added tags as long as 60bp with primers. In such a case I just ordered a 85bp oligo (HPLC purified) and no problem. I usually use Pfu for such PCR.

I too have used this method successfully and easily to introduce HA tags. Just one addition, you may need to do a ramp-up pcr in order to get efficient product. This is where you start with a low annealing and have it increase with each round. At first your primer will not anneal to the entire plasmid since the HA and restriction site are not complimentary but once you get a bit of product this becomes the new template which is complementary to the entire primer. Otherwise you can do a nested pcr where you take the product of the first pcr, which may not be all that much, and use it as the template in a second pcr. This should give you a much more robust reaction.

can try Phusion. Great enzyme. KOD long template is also another good enzyme.

If you are worried about the 10kb PCR reaction, look for a unique restriction site in your gene.
ie (Using NotI and BamHI as examples)

Plasmid----5'Gene ---- NotI-----Gene 3'---BamHI---Plasmid

Built a forward primer that binds to this unique restriction site within your gene.
And build a reverse primer as described by Ramses. (Although I would use 6bp as guard rather than 3bp)

PCR amplify and you will get
Guard---NotI------Gene3'-HA Tag-BamHI--Guard

Cut this PCR product with the appropriate restriction enzymes. Cut the plasmid with the same enzymes, remove the DNA fragment that contains the old 3' end of the gene. And then ligate them plasmid and PCR product together.

Plasmid----5'Gene ---- NotI-----Gene 3'-HA Tag---BamHI---Plasmid


Introduction

Genomic sequence variants may be inherited vertically (i.e., transmitted through the germline) or generated after zygote formation (i.e., leading to somatic or gonadal mosaicism). It is well established that somatic mosaicism occurs in cells of phenotypically normal individuals [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17] and can lead to various diseases [18]. However, the prevalence of somatic mosaicism and the extent to which it contributes to diseases outside of cancers requires elucidation [18].

Recent studies have estimated that each cell within the human brain contains hundreds to a few thousand somatic single-nucleotide variants (SNVs) and that a smaller fraction of cells harbor somatic copy number variations (CNVs) and mobile genetic element (i.e., retrotransposon) insertions [10, 15, 17, 19,20,21,22]. Dozens of somatic SNVs are present at high variant allele fractions (VAFs) across multiple tissues, indicating that they arose during early development [17, 23]. By comparison, some somatic SNVs are present at low VAFs and have limited tissue distributions, suggesting they arose later in development [15,16,17].

Single-cell DNA sequencing is the most direct approach to identify somatic variants. However, mutations introduced during the DNA amplification and/or generation of single-cell sequencing libraries, as well as non-uniform DNA amplification biases, make it difficult to discriminate bona fide mosaic SNVs from procedural artifacts [24]. Moreover, this approach for identifying mosaic SNVs requires sampling a large number of cells in a given individual and, consequently, is cost intensive.

Another approach to identify mosaic variants involves comparing bulk cell populations from two tissue samples derived from the same individual—the sample of interest and a control sample—as performed routinely during the analysis of cancer genomes. However, this approach is limited by the inability to define a proper control tissue because mosaic SNVs, particularly ones that arise during early development, are often present in multiple tissues across the body. Similarly, molecular barcoding approaches such as duplex sequencing can correct errors introduced by PCR amplification or sequencing and offer a > 10,000-fold improvement of accuracy when compared to conventional WGS [25, 26]. However, the most accurate molecular consensus approaches require extremely high sequencing depth (1000× or higher) to ensure that each DNA molecule is sequenced multiple times, thereby effectively utilizing only a few percent of the generated reads for variant calling [27]. From a practical standpoint, this requirement restricts the main benefit of barcoding to targeted approaches. Thus, the development of a unified set of best practices to detect somatic SNVs from bulk whole-genome sequencing (WGS) datasets would provide an alternative, cost-effective approach to identify somatic SNVs.

In this study, members of the Brain Somatic Mosaicism Network (BSMN) conducted a coordinated, multi-institutional study that analyzed mosaicism in a single neurotypical brain sample and established unified standards for calling and validating mosaic SNVs from bulk WGS and WES data.


HA-tag

Human influenza hemagglutinin (HA) is a surface glycoprotein required for the infectivity of the human influenza virus. The HA tag is derived from the HA-molecule corresponding to amino acids 98-106. It has been extensively used as a general epitope tag in expression vectors. Many recombinant proteins have been engineered to express the HA tag, which does not appear to interfere with the bioactivity or the biodistribution of the recombinant protein. This tag facilitates the detection, isolation, and purification of the protein of interest. [1]

The HA tag is not suitable for detection or purification of proteins from apoptotic cells since it is cleaved by Caspase-3 and / or Caspase-7 after its sequence DVPD, causing it to lose its immunoreactivity. [2] Labeling of endogenous proteins with HA-tag using CRISPR was recently accomplished in-vivo in differentiated neurons. [3]


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Other homology based technologies

We've described Sequence and Ligation Independent Cloning (SLIC) in a previous Plasmids 101 post. Although SLIC may be more cost effective, Gibson assembly improves on two aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease feature of T4 DNA polymerase, which must be controlled by the presence or absence of dNTPs. Secondly, in Gibson assembly a ligase is added to repair the nicks in vitro, whereas in SLIC these constructs are repaired in vivo, which ends up being much less efficient.

In addition to SLIC and Gibson, there are yet more homology based assembly methods that have been described--CPEC (circular polymerase extension cloning) and SLiCE (Seamless Ligation Cloning Extract) to name two more. Likewise, there are several store-bought cloning kits available which are all based on long-overlapping regions, have no requirements for restriction enzymes, and no scar sequences between fragments. Some of these products include:

Although these kits may come at a high price, their manufacturers tout great efficiency and low time commitments. These kits also come with specific protocols, suggestions for ratios of product to insert, and tools for primer design, so it’s always best to check in with the instructions from the particular manufacturer of the kit you will be using. Some of these products may offer advantages that Gibson assembly does not such as increased efficiency, shorter incubation times, or the ability to accommodate smaller fragments.


Contents

The development of methods to detect and identify biomolecules has been motivated by the ability to improve the study of molecular structure and interactions. Before the advent of fluorescent labeling, radioisotopes were used to detect and identify molecular compounds. Since then, safer methods have been developed that involve the use of fluorescent dyes or fluorescent proteins as tags or probes as a means to label and identify biomolecules. [3] Although fluorescent tagging in this regard has only been recently utilized, the discovery of fluorescence has been around for a much longer time.

Sir George Stokes developed the Stokes Law of Fluorescence in 1852 which states that the wavelength of fluorescence emission is greater than that of the exciting radiation. Richard Meyer then termed fluorophore in 1897 to describe a chemical group associated with fluorescence. Since then, Fluorescein was created as a fluorescent dye by Adolph von Baeyer in 1871 and the method of staining was developed and utilized with the development of fluorescence microscopy in 1911. [4]

Ethidium bromide and variants were developed in the 1950s, [4] and in 1994, fluorescent proteins or FPs were introduced. [5] Green fluorescent protein or GFP was discovered by Osamu Shimomura in the 1960s and was developed as a tracer molecule by Douglas Prasher in 1987. [6] FPs led to a breakthrough of live cell imaging with the ability to selectively tag genetic protein regions and observe protein functions and mechanisms. [5] For this breakthrough, Shimomura was awarded the Nobel Prize in 2008. [7]

New methods for tracking biomolecules have been developed including the use of colorimetric biosensors, photochromic compounds, biomaterials, and electrochemical sensors. Fluorescent labeling is also a common method in which applications have expanded to enzymatic labeling, chemical labeling, protein labeling, and genetic labeling. [1]

There are currently several labeling methods for tracking biomolecules. Some of the methods include the following.

Isotope markers Edit

Common species that isotope markers are used for include proteins. In this case, amino acids with stable isotopes of either carbon, nitrogen, or hydrogen are incorporated into polypeptide sequences. [8] These polypeptides are then put through mass spectrometry. Because of the exact defined change that these isotopes incur on the peptides, it is possible to tell through the spectrometry graph which peptides contained the isotopes. By doing so, one can extract the protein of interest from several others in a group. Isotopic compounds play an important role as photochromes, described below.

Colorimetric biosensors Edit

Biosensors are attached to a substance of interest. Normally, this substance would not be able to absorb light, but with the attached biosensor, light can be absorbed and emitted on a spectrophotometer. [9] Additionally, biosensors that are fluorescent can be viewed with the naked eye. Some fluorescent biosensors also have the ability to change color in changing environments (ex: from blue to red). A researcher would be able to inspect and get data about the surrounding environment based on what color he or she could see visibly from the biosensor-molecule hybrid species. [10]

Colorimetric assays are normally used to determine how much concentration of one species there is relative to another. [9]

Photochromic compounds Edit

Photochromic compounds have the ability to switch between a range or variety of colors. Their ability to display different colors lies in how they absorb light. Different isomeric manifestations of the molecule absorbs different wavelengths of light, so that each isomeric species can display a different color based on its absorption. These include photoswitchable compounds, which are proteins that can switch from a non-fluorescent state to that of a fluorescent one given a certain environment. [11]

The most common organic molecule to be used as a photochrome is diarylethene. [12] Other examples of photoswitchable proteins include PADRON-C, rs-FastLIME-s and bs-DRONPA-s, which can be used in plant and mammalian cells alike to watch cells move into different environments. [11]

Biomaterials Edit

Fluorescent biomaterials are a possible way of using external factors to observe a pathway more visibly. The method involves fluorescently labeling peptide molecules that would alter an organism's natural pathway. When this peptide is inserted into the organism's cell, it can induce a different reaction. This method can be used, for example to treat a patient and then visibly see the treatment's outcome. [13]

Electrochemical sensors Edit

Electrochemical sensors can used for label-free sensing of biomolecules. They detect changes and measure current between a probed metal electrode and an electrolyte containing the target analyte. A known potential to the electrode is then applied from a feedback current and the resulting current can be measured. For example, one technique using electrochemical sensing includes slowly raising the voltage causing chemical species at the electrode to be oxidized or reduced. Cell current vs voltage is plotted which can ultimately identify the quantity of chemical species consumed or produced at the electrode. [14] Fluorescent tags can be used in conjunction with electrochemical sensors for ease of detection in a biological system.

Fluorescent labels Edit

Of the various methods of labeling biomolecules, fluorescent labels are advantageous in that they are highly sensitive even at low concentration and non-destructive to the target molecule folding and function. [1]

Green fluorescent protein is a naturally occurring fluorescent protein from the jellyfish Aequorea victoria that is widely used to tag proteins of interest. GFP emits a photon in the green region of the light spectrum when excited by the absorption of light. The chromophore consists of an oxidized tripeptide -Ser^65-Tyr^66-Gly^67 located within a β barrel. GFP catalyzes the oxidation and only requires molecular oxygen. GFP has been modified by changing the wavelength of light absorbed to include other colors of fluorescence. YFP or yellow fluorescent protein, BFP or blue fluorescent protein, and CFP or cyan fluorescent protein are examples of GFP variants. These variants are produced by the genetic engineering of the GFP gene. [15]

Synthetic fluorescent probes can also be used as fluorescent labels. Advantages of these labels include a smaller size with more variety in color. They can be used to tag proteins of interest more selectively by various methods including chemical recognition-based labeling, such as utilizing metal-chelating peptide tags, and biological recognition-based labeling utilizing enzymatic reactions. [16] However, despite their wide array of excitation and emission wavelengths as well as better stability, synthetic probes tend to be toxic to the cell and so are not generally used in cell imaging studies. [1]

Fluorescent labels can be hybridized to mRNA to help visualize interaction and activity, such as mRNA localization. An antisense strand labeled with the fluorescent probe is attached to a single mRNA strand, and can then be viewed during cell development to see the movement of mRNA within the cell. [17]

Fluorogenic labels Edit

A fluorogen is a ligand (fluorogenic ligand) which is not itself fluorescent, but when it is bound by a specific protein or RNA structure becomes fluorescent. [18]

For instance, FAST is a variant of photoactive yellow protein which was engineered to bind chemical mimics of the GFP tripeptide chromophore. [19] Likewise, the spinach aptamer is an engineered RNA sequence which can bind GFP chromophore chemical mimics, thereby conferring conditional and reversible fluorescence on RNA molecules containing the sequence. [20]


Introduction

An unusually large number of mumps cases were reported in the United States in 2016 and 2017, despite high rates of vaccination [1,2]. In the prevaccination era, mumps was a routine childhood disease, with over 150,000 cases reported in the US annually [1]. After the mumps vaccine was introduced in 1967, mumps incidence declined by more than 99% [1]. Case counts rose again briefly in the mid-1980s and then continued to decrease after a national outbreak of measles prompted the recommendation of 2 Measles-Mumps-Rubella (MMR) vaccine doses in 1989 [3]. In the early 2000s, only a few hundred cases of mumps were reported annually in the US [1], attesting to the success of vaccination, possibly combined with decreasing clinical suspicion. This apparently low nationwide incidence was interrupted by an outbreak of >5,000 cases in the Midwestern US in 2006 [4], followed by a period of low incidence with minor outbreaks until 2016. This recent resurgence in mumps is partially explained by waning vaccine-induced immunity [5], but the extent to which genetic changes in circulating viruses have contributed is not yet clear.

In Massachusetts, over 250 cases were reported in 2016 and over 170 in 2017, far exceeding the usual state incidence of <10 cases per year [6]. As seen in other recent outbreaks, most cases were associated with academic institutions [4] and other close-contact settings, including prisons [7] and tightly-knit ethnic and religious communities [8,9]. Mumps was reported to the Massachusetts Department of Public Health (MDPH) by 18 colleges and universities in the state, including Harvard University (Harvard), University of Massachusetts Amherst (UMass), and Boston University (BU)—the 3 institutions with the largest numbers of reported cases. Of the individuals infected, 65% had the recommended 2 doses of the MMR vaccine (S1 Table).

We used whole genome sequencing, phylogenetic analysis, and transmission reconstruction to investigate the spread of mumps at multiple geographic scales, including within a college campus, more widely in Massachusetts, and across the US. Pathogen sequence data have become an important tool for understanding the spread of infectious diseases in near real time, allowing researchers to pinpoint outbreak origins [10,11], resolve transmission patterns [12], and detect changes throughout the genome that could affect disease severity or the effectiveness of vaccines and diagnostics [13–16]. Such data have been shown to be most useful when analyzed alongside epidemiological data [12,17,18], although the field is still exploring in detail how genomics can contribute to understanding and controlling outbreaks [19]. Mumps outbreaks in 2016 and 2017 in the US, particularly those in universities, provided an opportunity to apply these ideas to the mumps virus and to further this exploration in the context of a closely monitored, largely self-contained campus setting.


Designing PCR Primers for cloning - (Apr/12/2013 )

Hello,
Urgently need some help on a question for an assignment that has had me stumped for 2 days.

I'm supposed to clone and express a gene that encodes a protein/enzyme known as CobT produced by the bacterium Mycobacterium avium paratuberculosis. Now I need to design a primer to help me amplify, clone and express the protein. Problem is I'm totally confused on how to go about this.

How do you design a primer? I know this is a super basic question but the multitude of tutorials I've seen have me all confused.
I tried using the ncbi primer blast tool but it seemed all the primers it gave me were inside my target sequence which makes no sense, how do you use the ncbi tool to pick primers?

Is there a difference between a primer for cloning and one for PCR?

Any help would be appreciated.

The CobT nucleotide sequence is attached below, the target sequence is highlighted in blue.

OK as you want the full coding sequence of the gene - you have no option about where you put the primers, which is why primer BLAST wasn't working for you. You need to use the first 20-25 bp of the gene and the reverse complement of the last 20-25 bp of the gene, including the start and stop codons respectively. Try and match the GC content and length to give similar annealing temperatures for the primers, but it isn't essential.

If you need to add restriction sites or anything else to the primers, these always go on the 5' end of the primer. If you are doing this you also need to add a few (usually 6) bp to the 5' end before the restriction site so that the RE has somewhere to bind.

Essentially there is no difference between primers for cloning and primers for ordinary PCR, other than that cloning primers frequently have tails added that contain restriction sites or tags (for the protein, e.g. flag tag) added. These aren't essential if you are doing TA cloning or blunt end cloning.

bob1 on Sat Apr 13 00:08:45 2013 said:

OK as you want the full coding sequence of the gene - you have no option about where you put the primers, which is why primer BLAST wasn't working for you. You need to use the first 20-25 bp of the gene and the reverse complement of the last 20-25 bp of the gene, including the start and stop codons respectively. Try and match the GC content and length to give similar annealing temperatures for the primers, but it isn't essential.

If you need to add restriction sites or anything else to the primers, these always go on the 5' end of the primer. If you are doing this you also need to add a few (usually 6) bp to the 5' end before the restriction site so that the RE has somewhere to bind.

Essentially there is no difference between primers for cloning and primers for ordinary PCR, other than that cloning primers frequently have tails added that contain restriction sites or tags (for the protein, e.g. flag tag) added. These aren't essential if you are doing TA cloning or blunt end cloning.

Thank you for the great answer. I have a few more questions just so I'm sure I understood what you wrote. Correct me if I'm wrong.

Say this was a DNA sequence and I wanted to amplify the region underlined & in bold:
GAT CAT AAA ACA TGC TTG TAT AAA GGA TGC TGC CAT GTT CCG TGA ACT GGA AGC GAA CAA TCT TGC GGT
ATA TCA GAA AAA GCC AAA GCT GAT TGC AGT GCT TCT TCA GCG TAA TGC TCA
GTT AAA AGC GAA GGT TGT

1. My forward primer would be: 5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'
where the blue bases are the extra bases to allow the restriction enzyme to bind, the red are the restriction site and the orange is the start codon.

2. Reverse primer would be: 5' ( CTAAGT CCATGG CTA) TGA GCA TAA CGC TGA AGA AGC ACT 3'
where the yellow are the extra bases, the green is the restriction site and the purple bases are the stop codon.


-When then would you use primer blast or any of those primer design tools?

-During a tutorial we were given a large sequence and told to pick primers to amplify a given region within the sequence. Now we were told we could pick primers from anywhere in the large sequence as long as it flanked our region of interest. Is this another way to pick primers? Wouldn't that lead to you amplifying more bases than are required such that when you cloned the PCR product into a vector and tried to express it you might get another protein product mixing in with the protein you were targeting or maybe even a different protein product?
I've also seen a tutorial where the primers are the bases just before the region of interest i.e blue is sequence of interets, red is the primer.
3' GAT CAT AAA ACA TGC (TTG TAT AAA GGA TGC TGC CAT GTT CCG TGA ACT GGA AGC GAA CAA TCT) 5'
5' CTA GTA TTT TGT ACG

-Is there a reason for putting primers at different locations even though it seems as though its counterproductive?

The primers look good to me. SOme people advocate putting a 3-6 bp between the restriction site and the start/stop codon, but it shouldn't be necessary.

taiju on Sat Apr 13 01:58:12 2013 said:

-When then would you use primer blast or any of those primer design tools?

-During a tutorial we were given a large sequence and told to pick primers to amplify a given region within the sequence. Now we were told we could pick primers from anywhere in the large sequence as long as it flanked our region of interest. Is this another way to pick primers? Wouldn't that lead to you amplifying more bases than are required such that when you cloned the PCR product into a vector and tried to express it you might get another protein product mixing in with the protein you were targeting or maybe even a different protein product?
I've also seen a tutorial where the primers are the bases just before the region of interest i.e blue is sequence of interets, red is the primer.
3' GAT CAT AAA ACA TGC (TTG TAT AAA GGA TGC TGC CAT GTT CCG TGA ACT GGA AGC GAA CAA TCT) 5'
5' CTA GTA TTT TGT ACG

-Is there a reason for putting primers at different locations even though it seems as though its counterproductive?

minor comments about your proposed primers:

the third triplet from the added bases of the reverse primer: you wrote "taa", i think you meant "tta"

you end both primers with a "t", it's good practice to end with a "g" or a "c" to clamp the primer.

mdfenko on Mon Apr 15 13:24:21 2013 said:

minor comments about your proposed primers:

the third triplet from the added bases of the reverse primer: you wrote "taa", i think you meant "tta"

you end both primers with a "t", it's good practice to end with a "g" or a "c" to clamp the primer.

Thanks, I hadn't spotted that.

About ending the primers with a "G" or "C". the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:

FROM:
5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'

TO:
5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC 3'

The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?

If your sequence starts in ATG then its not really necessary to add a start codon right?

How would you use a primer design tool in such a way that it put your primers where you needed them, like in my case I need primers at the beginning of my coding sequence for the CobT protein as I want to be able to express CobT.

Okay, so I just used this information to make primers for my actual project.
The right primer is okay.
The left is giving me issues with regards to GC content, its at 68-70%. Is there a way to lower this other than trying to change up the primers I use, my ending sequence is pretty much full of G and C bases. These are the last 36bp of my protein sequence:
CAG GCC GGT GTG TCC GAC CCG TCC GCT CAC CCG TGA

taiju on Mon Apr 15 14:52:43 2013 said:

About ending the primers with a "G" or "C". the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:

FROM:
5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'

TO:
5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC 3'

The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?

you can increase as well as decrease. also, you don't have to make the change with triads, you can change one base at a time so you could just remove the "tt" to get to the "g" or you could have added a "c" (or "cc") to the end to set up the clamp.

remember, you are extending from the end of the primer so you don't have to make changes in threes to maintain the coding sequence.

the necessary primer length takes into consideration only the actual primer sequence. the rest doesn't anneal.

mdfenko on Mon Apr 15 16:27:16 2013 said:

taiju on Mon Apr 15 14:52:43 2013 said:

About ending the primers with a "G" or "C". the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:

FROM:
5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'

TO:
5' ( GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC 3'

The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?

you can increase as well as decrease. also, you don't have to make the change with triads, you can change one base at a time so you could just remove the "tt" to get to the "g" or you could have added a "c" (or "cc") to the end to set up the clamp.

remember, you are extending from the end of the primer so you don't have to make changes in threes to maintain the coding sequence.

the necessary primer length takes into consideration only the actual primer sequence. the rest doesn't anneal.

You just answered a question I was about to ask, thank you as I was wondering about what happens to the rest of the bases you add but if they don't anneal how do you get an amplicon that has the restriction site and the start/stop codons, like if only the actual primer sequence anneals and is replicated and the additional bases are just kind of hanging how do they get into the subsequently produced PCR products.. I hope my question makes sense.

The primer ends get copied by the subsequent cycles of PCR, its just the initial cycles where there are "free" ends.


Additional file 1: Figure S1.

Arabidopsis and human assembly dotplots. Figure S2. M82 RaGOO confidence score distribution. Figure S3. Heinz cDNA alignment. Figure S4. Annotation Edit Distances. Figure S5. S. pennellii dotplots. Figure S6. S. pennellii confidence score distributions. Figure S7. A. thaliana pan-genome SV distribution. Table S1. Sequence statistics for simulated tomato genomes. Table S3. Performance statistics for Tomato chromosome construction.


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