PCR Product Sequencing Produkta PCR sekvenciranje
How To Sequence Your PCR Products Kako zaporedje vaših produktov PCR
Once you amplify your DNA sequence of interest, you usually need to check the amplification quality and accuracy using DNA sequencing services. Ko razširjajo vaše zaporedje DNA obresti, ki jo je ponavadi treba za preverjanje amplifikacije kakovost in točnost sekvenciranje DNK, ki uporabljajo storitev. There are several methods to do this and each method has its advantages and disadvantages. Obstaja več načinov, da to stori in vsaka metoda ima svoje prednosti in slabosti.
Direct Sequencing of PCR Products Neposredna zaporedja produktov PCR
Direct sequencing of PCR products is obviously the quickest method although it is not always the most successful method. Neposredna zaporedja produktov PCR je seveda najhitrejši način, čeprav ni vedno najbolj uspešen način. It is possible to directly sequence a PCR product without having first cloned the fragment into a sub-cloning vector such as a TOPO plasmid. To je mogoče pripisati neposredno zaporedje PCR izdelek brez prve klonirane je razdrobil v sub-kloniranje, kot je vektor plazmid TOPO. Obviously there are many advantages to the direct sequencing approach. Seveda obstajajo mnoge prednosti, ki neposredno sekvenciranje pristop. However, there are several disadvantages and steps which many people bypass only to have failed DNA sequencing results. Vendar pa obstajajo številne pomanjkljivosti in ukrepe, ki jih mnogo ljudi samo za obvoznico niso zaporedja DNK rezultate.
Direct sequencing of PCR fragments is hardly successful unless you learn how to make it successful. Neposredna zaporedje PCR fragmentov je težko uspešno, če se naučite, da bo uspešen. We have generated the following tips to make sure you don't fail in your direct sequencing attempts. Ki smo ga spodbudili naslednje nasvete, da poskrbite, da boste ne ne v vaši neposredni Zaporedje poskusov.
1) Make sure that you have the right PCR product before you send your PCR product for sequencing. 1) Poskrbite, da imate pravico produkta PCR, preden jo pošljete vaš produkta PCR za sekvenciranje.
With sub-cloning PCR products into a vector, people usually restriction enzyme digest the bands out of the vector to check if it is the right sequence. S sub-kloniranjem produktov PCR v vektorju, ljudje ponavadi omejitev encim prebavljiv pasovih iz vektorja, da preverite, če je pravica zaporedje. Make sure you check the size of your PCR product carefully on the gel before you send it for sequencing. Poskrbite, da vam preverijo velikosti vašega produkta PCR natančno na gelu, preden ste ga poslali na sekvenciranje. Also, if there are known restriction sites in the PCR fragment, make sure you use these to check that your product is the right one. Tudi, če obstajajo znane omejitve straneh v PCR fragmentov, poskrbite, da boste uporabo teh za preverjanje, da vaš izdelek je pravica enega.
2) Make sure you don't have many non-specific products (bands on agarose gel)! 2) Poskrbite, da ne boste imeli veliko nespecifične izdelke (pasove na agaroznem gelu)!
PCR reactions rarely produce only a single (one) band. PCR reakcije se le redko proizvajajo le en (en) pasu. Usually you will have many bands (non-specific products). Običajno boste morali veliko pasov (nespecifične izdelke). Even if you can't see them, your PCR reaction may have many bands (non-specific products) that will cause your DNA sequencing to have many signals at each nucleotide (you get a jumbled sequence ie N (all 4 nucleotides) instead of 1 nucleotide). Tudi če ne morete videti, vaše reakcije PCR lahko imajo mnogi pasovih (nespecifične izdelke), da bo povzročil vaš zaporedja DNK, da imajo mnoge signale na vsaki nukleotidov (dobite Ispremiješan tj zaporedje N (vseh 4 nukleotidov), namesto da 1 nukleotidov).
Using nested PCR primers can reduce this problem. Uporaba Vstavljena PCR primerji lahko zmanjšajo ta problem. It is also less likely that non-specific product will co-amplify through a second round of PCR with internally- Prav tako je manj verjetno, da je bila nediskriminatorna poseben izdelek bo co-razširjajo prek drugega kroga PCR z notranjo -
nested primers. Vstavljena oligonukleotidov.
3) Remove all residual PCR primers and unincorporated nucleotides before sequencing. 3) odstrani vsa preostala PCR primerji in nekorporativnih pred sekvenciranje nukleotidov.
The good thing with sub-cloning is that you clone in the PCR fragment into the vector, amplify it, and purify it. Dobra stvar pri sub-kloniranje je, da si klon v PCR fragmentov v vektorju, razširjajo, in jo Prečistimo. This way you have a very clean batch of DNA. Na ta način imate zelo čist serije DNK. After PCR amplification however, you have a lot of unincorporated nucleotides, pcr primers, salts and proteins that can interfere with DNA sequencing. Po PCR ojačanja pa imate veliko nekorporativnih nukleotidov, PCR primerji, soli in proteinov, ki lahko motijo delovanje zaporedja DNK. Make sure you at least use a PCR clean-up step to ensure your DNA sequencing doesn't fail. Poskrbite, da boste vsaj uporabo PCR čiščenje korake, da zagotovita vaš zaporedja DNK, ne, ne.
4) Ensure you don't send a very concentrated DNA sample! 4) Zagotoviti, ne boste poslali zelo koncentrirana DNA vzorec!
Make sure that you double-check the template DNA concentrations before you send your PCR for sequencing. Prepričajte se, da ti dvojno preverjanje matrice DNK koncentracij, preden jo pošljete vaš PCR za sekvenciranje. This is because PCR fragments are smaller than plasmids, and thus you get more DNA per sample. To je zato, ker so manjše fragmente PCR kot plazmidi, in tako dobiš več DNA na vzorec. This makes PCR products more effective sequencing templates than the usual plasmids. To pomeni, da produktov PCR učinkovitejše Zaporedje predloge, kot je običajno plazmidov. Therefore you need to have lower concentrations for PCR products. Zato boste potrebovali, da so nižje koncentracije za produktov PCR.
For example, a 200 bp fragments needs to be just 2 ng/ul! Na primer, 200 bp fragmenti treba samo 2 ng / ul! If your samples are at too high a concentration, not only will they NOT sequence any better but may cause your DNA sequencing to fail. Če je vaš vzorci so tudi na visoki koncentraciji, ne le ti ne bo nič bolje, ampak zaporedje lahko povzroči zaporedja DNK, da bi doživelo neuspeh. Estimate your PCR product concentration by loading them with standards on an agarose gel. Ocenite vašega produkta PCR koncentracije z nakladanjem jim s standardi na agaroznem gelu. Usually laboratory spectrophotometers cannot with accurately measure the small amount of DNA that a PCR reaction generates (unless you use the newer micro-specs ie "Nanodrop"). Običajno laboratorijsko spectrophotometers ne more natančno izmeriti z majhno količino DNA, da se ustvari reakcije PCR (razen če uporabljate novejše mikro-specs tj. "Nanodrop"). Based on your analysis, make sure you dilute them. Glede na vašo analizo, se prepričajte, da ste jih razredčimo.
Subcloning PCR Products and Sequencing them Subcloning Produkti PCR, in jim Sekvenciranje
The most effective way to sequence PCR products is by subcloning them into vectors or plasmids. Najbolj učinkovit način za zaporedja produktov PCR je z njimi v subcloning vektorji ali plazmidi. Usually this was done with primers that contained restriction enzyme sites. Ponavadi je bilo to storjeno z oligonukleotidov, ki bi vseboval omejitev encim straneh. There were many problems with this including the fact that restriction enzymes have difficulties cutting at the ends of a PCR fragment. Je bilo veliko težav s tem, vključno z dejstvom, da omejitev encimi imajo težave rezanje na koncih fragmentov PCR.
After cutting the PCR fragment, one would have to ligate the PCR fragment into a pre-cut vector that has compatible ends. Po razseku PCR fragmentov, ena bi morala ligate PCR fragmentov v vnaprej narezanem vektor, ki je združljiva konča. After amplification and purification of the plasmid, one would simply send the fragment for DNA sequencing. Po ojačanja in očiščevanje z plazmid, ena bi preprosto pošlje fragmenta za sekvenciranje DNK. Most vectors have T7, T3, M13, and other primer sites which allows easy DNA sequencing using universal primers (such as the commonly available T7 primer, T3 primer, and M13 primers). Večina vektorji so T7, T3, M13, in primer drugih spletnih strani, ki omogoča enostavno sekvenciranje DNK, ki uporabljajo univerzalne oligonukleotidov (kot je običajno na voljo T7 temeljni premaz, temeljni premaz za T3 in M13 oligonukleotidov).
TOPO Vectors for PCR sub-cloning TOPO Vectors za PCR sub-kloniranje
The latest greatest innovations allowed many to bypass the difficulties or subcloning PCR products into regular vectors. Najnovejše inovacije največje dovoljeno veliko, da se izogne težavam ali subcloning produktov PCR v rednih prenašalcev. TOPO cloning allowed the RAPID and easy cloning of PCR products into vectors. TOPO kloniranje dovoljeno hitro in enostavno kloniranjem produktov PCR v vektorje. TOPO cloning is very quick (5 minutes for ligation) and very effective. TOPO kloniranja je zelo hitro (5 minut za ligation) in zelo učinkoviti. You can easily clone your PCR fragment into the vector without any restriction sites. Z lahkoto lahko klon vaš PCR fragmentov v vektor brez kakršnih koli omejitev straneh. This is due to the fact that many DNA polymerases add an extra A to the ends of the DNA fragment. To je posledica dejstva, da je veliko DNA-polimeraze, dodalo dodatno A do krajev, fragmentom DNA. This allowed the cloning of the PCR fragments into TOPO vectors (or TA vectors) which contained free T ends. To je omogočilo kloniranje PCR fragmentov v TOPO prenašalcev (vektorjev ali TA), ki je vsebovala proste T konča. TOPO vectors contain Topoisomerase enzyme which enhances the cloning of PCR fragments into the vector. TOPO vektorji vsebujejo topoizomeraze encima, ki spodbuja kloniranje PCR fragmentov v vektor.
TOPO vectors are available at Invitrogen TOPO vektorji so na voljo pri Invitrogen
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