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Protocol #2: Agarose gels.

image

The following protocol is for making and running an agarose gel for DNA. Very easy to do and one of my favorite procedures besides immunofluorescence (which I may post later). 

Materials:

  • Powdered agarose
  • TE buffer (TAE or TBE)
  • Flask
  • Microwave
  • Casting tray (varies with each lab)
  • Gel comb with appropriate number of teeth
  • DNA samples 
  • Loading dye
  • Electrophoresis chamber and power supply
  • Ethidium bromide 

Directions:

  1. To prepare the gel, add the appropriate amount of TE buffer to powdered agarose in a flask with plenty of empty space. To make a 1% agarose gel (standard concentration), add 100 mL TE to 1 mg agarose. Higher agarose concentrations (up to 2%) are typically used for small DNA fragments, while lower concentrations (.7%) are for larger DNA fragments. 
  2. Heat liquid agarose (agarose in TE) in the microwave until solution starts to boil. WHILE WEARING GLOVES, remove flask from microwave and swirl gently. Look for chunks of agarose and/or remaining powder. 
  3. Reheat in the microwave until solution starts to boil again. At this point, swirl again. The solution should be devoid of chunks or any powdered agarose. If they are present, heat again until the solution appears clear.
  4. Let flask cool until it can be handled by (gloved) hands. In the meantime, set up casting tray, making sure that the tray is LEVEL. If needed, use a leveling bubble to check. Insert gel comb in the notches closest to the edge of the casting tray. 
  5. Once the flask has cooled, add ethidium bromide in a 1 uL: 25 mL concentration. Too much ethidium bromide may cause the entire gel to fluoresce and be difficult to image, while not enough ethidium bromide wouldn’t allow the DNA to bind properly and fluoresce. 
  6. Pour agarose into casting tray SLOWLY as to prevent bubbles. Pour gel until it reaches halfway up the comb. Let gel solidify completely. The gel is solidified when it appears a milky white. 
  7. Prepare DNA samples. Depending on the purpose for electrophoresis, you may need to change sample volumes in order to have a standard mass of DNA. 
  8. Add loading dye to each sample. Loading dye is primarily used to visualize electrophoresis and allow DNA to stay in the well, so how much you add is variable. 1 uL is a good standard to go by. 
  9. Once gel has solidified, place gel in electrophoresis chamber and add TE buffer over it. Pour TE buffer until it covers the gel and remove gel comb. If the wells are sticking out over the surface of the buffer, add more buffer until they are completely covered. 
  10. Carefully add samples to wells, making sure to save one well for ladder. Try to avoid sample leaking out of the wells, as they could leak into other wells and affect results. 
  11. Cover electrophoresis chamber and plug in power supply. Turn power supply and set voltage (I use 80-125 volts, depending on expected fragment size and time desired). Start gel running. Check that gel is indeed running by looking at current function on the power supply or looking into the chamber to see if there are bubbles at each end. 
  12. Check to see that loading dye separates into two different bands (colors may vary depending on type of loading dye used). 
  13. Let gel run depending on expected fragment size. If fragments are small (100-200 bp), run only halfway down the gel. If fragments are larger, let the dye run until ~2 cm from the opposite edge of the gel. 
  14. Turn off power supply and carefully remove gel (which may slide). Visualize gel with a BioRad imager or transilluminator. Be careful when using transilluminator - UV irradiation may cause sunburn and/or DNA damage to skin. Always view gel with protective glass between you and the gel. If you need to touch the gel, make sure hands and arms are covered and ALWAYS protect eyes from UV exposure. 

Hope this is helpful to anyone who needs it! As usual, ask questions about the protocol or leave comments! 

‘Dirt cheap’ Seaweed Bio-Nano-Chip

futurity.org

Microsponges derived from seaweed are a key component of a tiny programmable chip designed to sniff out diseases such as HIV and cancer.

The microsponges are 280-micrometer beads of agarose, a cheap, common, lab-friendly material made from seaweed and often used as a matrix for growing live cells or capturing proteins.

Click through to read more and to view video from Rice University.  

Not surprisingly, the ‘agarose’ tag is pretty damn lacking.

Odd

I have done quite a few Gel-Extractions in the past few years (all with NaI and silica gel) but for some reason I cannot get it to work with the Promega columns and RNA-Seq libraries. I had the same source RNA (10ug) which I size selected once with Ampure XP beads and once with a 2% agarose gel (I had run out of Ampure beads). With the ampure beads I ended up with just barely enough product but at the “correct” size but with gel extraction all I got were primer dimers (<100bp so not adaptor dimers). The other time I tried gel extraction for size selection I also had primer dimers but I figured that was a fluke. Other people have great results with the agarose gel extraction but not me. 

Take Home message… don’t keep trying something if it doesn’t work in your lab

Procedimento Operacional Padrão - Eletroforese

1 Objetivo

É objetivo deste POP padronizar metodologia de eletroforese em gel de agarose para separação de moléculas, envolvendo a migração de partículas quando da aplicação de uma diferença potencial.

2 Campo de aplicação

Este procedimento aplica-se a disciplina de estágio supervisionado de biotecnologia.

3 Descrição

3.1 Materiais utilizados

a) tampão de eletroforese (Tris-acetato-EDTA - TAE ou Tris-borato-EDTA - TBE);

b) tampão de corrida (Migração: azul de bromofenol - 300bp e xileno cianol - 4000bp);

c) brometo de etídeo;

3.2 Equipamentos e utensílios

a) cuba de eletroforese;

b) fonte de força;

c) bandejas de gel (diferentes tamanhos e de plástico transparente à UV);

d) pentes (para fazer poços);

e) transiluminador;

f) pipetas de precisão;

g) béqueres de 500 mL;

h) erlenmeyers de 500 mL;

i) pipetador automático;

4 Metodologia

4.1 Preparo de gel de agarose (0,8%) 

a) Para 100 mL de gel, pesar:

agarose……………………8 g

b) adicionar 100mL de TAE 1X;

c) fundir a solução no microondas até homogeneizar (aproximadamente 1 min e 10 seg na potência máxima – evitar fervura);

d) enquanto a solução de gel esfria (morno), preparar o suporte de gel, selando as laterais com fita crepe ou na própria cuba;

e) acrescentar 4µL de Brometo de Etídeo (10mg/mL) e misturar com agitação leve;

obs.: brometo de etídio é mutagênico e deve ser manuseado com luvas.

f) despejar a solução de gel no suporte sem fazer bolhas;

g) colocar o pente da espessura desejada no suporte;

obs.: verificar o número e o volume das amostras para escolher o pente mais adequado. 

h) esperar a solução solidificar;

i) colocar o suporte com o gel na cuba de eletroforese;

j) adicionar tampão TAE 1X até cobrir a superfície do gel.

4.2 Analítica

k) extrair 3µL da amostra de DNA;

l) homogeinizar com 1µL de tampão de corrida 5X (Loading Buffer);

m) aplicar as amostras nos poços;

n) reservar 2 poços para utilização de um padrão de corrida (Ladder-1Kb);

o) ajustar a voltagem de acordo com o tempo de corrida desejado (média utilizada 80V);

p) analisar o gel sob radiação ultravioleta (Alpha Imager);

q) tirar foto dos resultados obtidos;

4.3 Preparativa

r) adicionar tampão de corrida ao material a ser purificado (diluição de 5X);

s) aplicar as amostras nos poços, pulando um poço entre cada amostra para evitar contaminação;

obs.: para volumes maiores, montar o gel com o pente de dentes largos ou unir os dentes do pente com fita adesiva antes de colocar o pente no gel ainda líquido (morno).

t) aplicar em outro poço o padrão de tamanho molecular (Ladder-1kb);

u) ajustar a voltagem de acordo com o tempo de corrida desejado (média utilizada 80V);

v) vizualizar as bandas no gel utilizando a luz ultravioleta manual no comprimento de onda longo;

w) cortar a banda desejada utilizando bisturi limpo e colocá-la em um tubo de microcentrífuga previamente pesado;

x) seguir protocolo de Purificação de DNA a partir de Gel. 

4.4 Soluções

y) Tampão de corrida 

Solução TAE 1X (Tampão Tris-Acetate - Tris 40mM, Ac. Acetico 20mM,EDTA 1mM) 

diluir a solução estoque 50x em água Milli-Q. 

z) TAE 50X 

(Solução estoque) p/ 1L 

Trizma-Base     242,0g 

Ácido acético glacial     57,1mL 

EDTA 0.5M pH 8.0     100,0mL 

água q.s.p.     1000,0mL 

aa) Tampão de amostra 5X 

(Loading Buffer) p/ 50mL 

Glicerol (50%)     25,0mL 

Azul de Bromofenol (0.125%)   0,0625g 

Xileno Cianol (0.125%)     0,0625g 

TE pH8,0 q.s.p.     50,0mL 

bb) Padrão de tamanho molecular 

(Ladder-1kb) (0,1µg/µL)

Ladder 1kb (Gibco BRL - 1µg/µL)   100µL 

Tampão (Tris 10mM pH7,5, NaCl 50mM, EDTA 0,1mM)   500µL 

Loading Buffer 5X     400µL

cc) Brometo de etídeo (10mg/mL) 

Brometo de etídeo     1g 

Água Milli-Q q.s.p.     100mL 

Agitar durante 16 horas, guardar a 4°C. 

obs.: concentração no gel: 5µg/mL

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