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24 marzo 2009 - 19:59

Disegnare velocemente Primer con CLC…

A seconda del tipo di PCR che si deve fare (Real Time Pcr o no, da genomico o da plasmide) si devono costruire primer più o meno “accurati”. Se si deve fare una Pcr da genomico ad esempio bisogna stare attenti che i primer non vadano anche ad annilare in altri punti del genoma, oltre a quello di interesse…

Nel mio caso faccio molte PCR da plasmidi. In questo caso le coppie di basi non sono molte, quindi è altamente improbabile che primer lunghi 15-20 nucleotidi vadano ad annilare in più punti.

In questo caso disegnare dei primer è molto semplice…Ma troppe volte vedo persone che disegnano su carta i primer, sia forward che reverse (perdendo tempo e rischiando di fare errori).

La cosa è molto più semplice con CLC Sequence Viewer:

  • (Ovviamente) Importare la sequenza del gene su cui fare la PCR in CLC Sequence Viewer
  • Selezionare lo spaziamento ogni 3 nucleotidi scegliendo il frame appropiato (Potete anche salvare questa opzione in modo da non selezionarla ogni volta).

schermata-clc-sequence-viewer-save-setting1

  • Selezionare i 15-20 nucleotidi che formeranno il primer FW (tenendo conto del frame e che devono terminare con una C o G), poi tasto dx -> Open Selection in new view
  • Tasto dx sul primer appena creato -> Select Sequence, di nuovo tasto dx -> Edit. In questo modo potete modificare la sequenza, aggiungere codoni di start, enzimi di restrizione.
  • Tornando sulla sequenza del gene selezionate il segmento che andrà a formare il primer Reverse. In questo caso non c’è bisogno di tenere conto del frame. Fate in modo che cominci con una G o una C (l’inizio che selezionate diventerà il 3?, quindi rappresenta la fine del primer). Al solito, fate tasto dx -> Open Selection in new view. Poi modificate il primer a vostro piacere aggiungendo codone di STOP o siti di restrizione.
  • A questo punto nella toolbox “Reverse Complemente Sequence” e avrete il vostro primer Reverse pronto.

schermata-clc-sequence-viewer-toolbox

I primer possono essere copiati-incollati sul sito dove ordinate i primer (evitate errori di battitura), possono essere stampati o salvati in una cartella. Vi ricordo che se si deve fare una Pcr da genomico bisogna stare attenti che i primer non vadano anche ad annilare in altri punti del genoma, quindi questo metodo non va bene (in questo caso c’è il sistema descritto da Nico).

Spero però sia utile a qualcuno!!

Alla prossima…

Link: MyBioinformatica

Tags: bioinformatica, how-to
9 dicembre 2008 - 09:00

How to Get Rid of Fleas in Your House, in Your Yard, and More

Fleas are some of the most annoying pests to deal with. They’re small, jumpy, and multiply quickly. Pets can pick up fleas from being outside in nature, around other animals, or when humans track in the insects on our shoes or clothes.

Excessive itching and scratching is a telltale sign your pet may have fleas. You may even spot the little acrobats. On average, fleas are 2-4 millimeters long, making them visible to the naked eye.

If your family pet has fleas, it’s likely that your yard and house will become a breeding ground. Acting quickly is key to preventing spread.

A flea infestation can test your patience and require persistence. But you can eradicate the problem with a combination of cleaning methods, sprays, and pet-friendly topical medications among other options.

Do fleas bite people, too?

Yes! Fleas are after blood and can bite people or latch onto our clothes or shoes. However, they really do prefer animals. Pets are ideal hosts because thick fur provides plenty of shelter for fleas to latch onto skin and feed, or lay eggs.

If fleas do bite you during an infestation, it will likely be around your ankles, or in folds of skin. Flea bites can cause an allergic reaction in the form of hives.

 

According to the Centers for Disease Control, the life cycle of the fleaTrusted Source depends on a lot on environmental conditions. Fleas flourish in warm climates and usually (but not always) die in the winter season.

Flea eggs are small but can be seen if you’re looking closely. They are smooth and white or light in color. A single adult female flea can produce up to 2,000 eggs in her lifetime. These eggs may be laid in your pet’s fur, deep in the carpet, or in tall grass.

In ideal conditions, fleas will evolve from egg to adult within 2 to 3 weeks. Adult fleas can live up to 100 days.

Fleas are ready to feed within a day of hatching, and begin to suck blood within 10 seconds of landing on a host.

If the weather isn’t ideal and there isn’t a host to feed on, flea larvae may remain dormant for months while waiting for better conditions to develop. This is a key reason dealing with infestations can be so difficult.

How long does it take to get rid of fleas?

The length of time required to get rid of a flea infestation depends on your environment, and how long the fleas have been there.

Prompt cleaning and using topical flea medications for your pet may get rid of the majority of fleas within a day or two. However, it can take days to weeks for all the fleas present in an environment to die, even with the most conscientious approach.

Fleas lay a lot of eggs very quickly, and some fleas have developed resistance to medications and insecticides. If you have a large property or multiple pets with fleas, it may take longer to get rid of the problem. The key to handling fleas is persistence.

How to get rid of fleas in your home

If your pet is a walking carrier of mature fleas, your home can become the nursery.

Since the flea has multiple life stages (egg, larvae, cocoon, adult), when adult fleas are present, it is assumed all of these stages are also present throughout your house. This means that you have to tackle the problem from all angles in order to truly eradicate the infestation. Learn more detailed information from this flea and tick prevention for dogs article.

To do this, you must treat your pet and its living environment at the same time. Depending on your pet’s boundaries, this may include your whole house or yard.

The Environmental Protection Agency (EPA) recommends the following cleaning approaches:

  1. Use a powerful vacuum on any floors, upholstery, and mattresses. Cracks and other tight spaces are usually good hiding places for fleas and their cohort of eggs, larvae, and cocoons. If you can, use a vacuum with a bag you can dispose of without coming into contact with its contents.
  2. Employ a steam cleaner for carpets and upholstery, including pet beds. The combination of high heat and soap is the enemy of fleas in all stages of life. Pay special attention to any spots where your pet usually lies down or spends a lot of time.
  3. Wash all bedding, including your pet’s, in hot water and detergent. Dry it at the highest heat setting. If the infestation is severe, consider getting rid of old bedding and starting anew.

The advent of topical flea treatmentsTrusted Source for pets have made insecticides pretty outdated. Topical prescriptions stop or augment the flea’s reproductive cycle and rapidly kill an infestation.

If you do aim to use an insecticide or other chemical cleaning treatment, please proceed with caution. Many are toxic to humans, pets, and the environment.

Here are some tips:

  • Aerosol sprays are recommended over foggers, as you can direct the spray under beds or other places that the foggers may be unable to reach.
  • Choose an insecticide that contains both an adulticide (kills adult fleas), such as permethrin, and an insect growth regulator (kills the eggs, larvae, and pupae), such as methoprene or pyriproxyfen.
  • People and pets shouldn’t come into contact with an insecticide or chemical treatment until it has dried. Be sure to wear gloves when you apply the spray, and only do it when everyone is out of the room or house.
How to get rid of fleas in your yard

The best way to eliminate fleas from your yard is to think about where they’re most like to hide.

Fleas love places that are:

  • shaded
  • humid
  • warm

Direct sun-exposed areas can get too hot, so you probably won’t find many fleas there. Problem areas will likely be found by observing where your pet likes to lie down.

Once you have your target zones, here’s what you can do to eliminate the fleas:

  1. Mow your lawn regularly and rake the exposed surfaces thoroughly. Fleas like to hide in tall grass. Make sure to bag the contents rather than add them to your compost pile.
  2. Remove debris, such as dead leaves and twigs, from flower beds and from under any bushes. Expose as much of the shady areas to sunlight as you can.
  3. Spread cedar chips on the areas where your pet likes to lie down, under the bushes, and on flower beds. Fleas hate the smell! Sulphur (powder or liquid) is also known to repel fleas and prevent hatching.
  4. Ask your local gardening center about nematodes, small worms that can eat insect larvae.
  5. Avoid overwatering. This can create the exact humid conditions for fleas to thrive in.
  6. Evict wildlife. Animals like opossum, mice, and squirrels can all carry fleas. It’s possible to repel these animals from your yard without trapping or killing them. The Humane Society recommends “gently harassing” animals to get them to move. This can include setting up barriers in the yard, putting up bright lights, playing loud music, and leaving rags soaked in cider vinegar.
Tags: bioinformatica, how-to