Silvia♥

[Silvia♥ @silviia_08]

No me dejes

[Bento Lab 🧬 @theBentoLab]

For anyone extracting PCR products from agarose gels (or who would like the option to do so occasionally), here’s a cheap and rapid homemade spin-column gel extraction method that’s reportedly good enough for Sanger sequencing without further purification.👇

The method, described by Abraham et al. (2017), is based on the observation that low concentrations of TAE (Tris-acetic acid-EDTA) buffer do not seem to inhibit PCR and Sanger sequencing. This means that a quick centrifugation of a gel slice through a filter could be enough to produce sequencing-ready DNA.

The authors made homemade spin columns by placing a small amount of cotton fiber (moistened with 0.5x TAE buffer), in a 0.5 mL centrifuge tube, and poked a hole in the bottom of the tube. This tube was then placed inside a 1.5 mL tube, and was then ready for use.

An electrophoresis gel was run using 0.5x TAE buffer, and successful amplicon bands cut out and placed in the homemade spin columns. The gel slices and columns were then centrifuged at 5200×g for 5 minutes to produce an extract used for Sanger sequencing reactions.

The authors reported a slight reduction in DNA yield compared to commercial gel extraction kits, but this didn’t impact results and their method was much cheaper and quicker. However, they did suggest that it’s best used for strong PCR bands, and it may not be quite as efficient as methods that totally remove TAE buffer. Their article can be found here:

Abraham et al. (2017). A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing. 3 Biotech, 7, 1-6.
https://t.co/TxeBRhbzql

Interestingly, this method appears to be a hybrid of the classic “freeze-and-squeeze” method (where the agarose is frozen before centrifugation to extract DNA), for example as described by Tautz & Renz (1983):

Tautz & Renz (1983). An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Analytical biochemistry, 132(1), 14-19.
https://t.co/K29Bf4n26S (subscription access).

… and Millipore’s Montage(TM) DNA Gel Extraction Kit, which uses proprietary filter columns plus a gel shredder to improve extraction efficiency without freezing. See Leonard et al. (1998) below for a description of the method using what is presumably a previous iteration of this kit:

Leonard et al. (1998). Preparation of PCR products for DNA sequencing. Biotechniques, 24(2), 314-317.
https://t.co/PyFdKTV3cD

Looking at all three protocols, two additional modifications to Abraham et al. (2017)'s method spring to mind:

⭐Replace the homemade spin column with a cut-down filter-pipette tip, making the method even cheaper and more accessible. You can find a great video by Rahul Patharkar on using filter-tip freeze-and-squeeze here: https://t.co/YypOhmbWtA !

⭐Reduce the EDTA concentration in the TAE buffer to 1/10 of its normal concentration, in-line with Millipore’s Montage(TM) DNA Gel Extraction Kit recommendations.

In other words, you could try running a gel in a low EDTA TAE buffer to visualise amplicons, cut out gel bands, and centrifuge through filter tips to extract a (hopefully) Sanger sequencing-ready DNA extract.

As a disclaimer, Abraham et al. (2017) and Leonard et al. (1998) both used ethidium bromide in their gels. Other DNA stains might (or might not) have more of an impact on sequencing quality.

Also, if you do try it, I'd suggest testing on only a few samples to begin with just in case of failures!

And if you already use this method, or something similar, we'd love to know how works for you!

https://t.co/TxeBRhbzql

[AlexMartiniDj @YoAlexMartini]

DEI V DEI V 😍

[Silvia♥ @silviia_08]

Podría ser yo perfectamente

[Anyone @anyxne]

He tenido un día muy intenso y he decidido afrontarlo de la manera más adulta posible.

[Chollometro @chollometro]

🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩🤩
OJO: Un cheque de 50 euros (para Amazon España) le va a tocar a una de las personas que decida darle a 🔁 a este tweet y seguir a Chollometro.

PD: Dale a ❤️ para que el poder del chollo te acompañe. El martes decimos ganador/a
🤩🤩🤩🤩

[Chollometro @chollometro]

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50 euros para gastar en amazon (España) van a ser para una de las personas que haga RT 🔁 y siga a Chollometro. Así de simple!

Dale mucho ❤️ a este tweet para que la suerte te acompañe. El jueves decimos ganador.

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[Chollometro @chollometro]

😱😱😱😱😱😱😱😱😱😱😱😱😱😱😱😱😱😱
VAMOS!!!!! 50 euros para gastar en amazon (España) van a ser para una de las personas que haga RT 🔁 y siga a Chollometro.

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[Chollometro @chollometro]

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Le van a tocar a 1 de las personas que haga 🔁 a este tweet y siga a Chollometro.

Dale mucho ❤️ 🔥

[Silvia♥ @silviia_08]

@alvaar00

[Silvia♥ @silviia_08]

Podría ser yo perfectamente

[Ilyas Alav @ilyas_alav]

So how is your Monday going? 🥲

[Wuolah @Wuolah]

hoy estoy un poco Tía Loli

[Ávila Escondida @AvilaEscondida]

#Nieve_en_Ávila: Aunque sólo lo sepan los pocos vecinos de este pueblecito de la Sierra de Ávila, también ha nevado en Marlín (Ávila) (1)

@Lavozabulense @CyLesVida @TurismoEnAvila @turismodeavila @Avila_CyL @avilatuitea @Turistaporavila @EnPueblo @ES_Despoblacion @dipuavila

[Elisa Granato @Prokaryota]

ending the day with a failed PCR

[maría 🌻 @mariasty_]

en el laboratorio: 87 pasos para preparar la muestra

esta gente: me la pela meto el trozaco de ferrero tal cual

[Silvia♥ @silviia_08]

Hacer R y sentirse inútil van de la mano 😵‍💫

[Informa Cosmos @InformaCosmos]

Caído pero no derrotado. https://t.co/euF4tad8uN

[AlexMartiniDj @YoAlexMartini]

QUEVEDO EDITION

[Salsichinhas @salsichinhas]

🌼🌼🌼🌼🌼🌼🌼🌼

[ceciarmy @ceciarmy]

Frank de la jungla ayudando a un mono a subir un árbol es lo mejor que he visto hoy

[NewsDigitales @NewsDigitalesAr]

quisiera estar tan relajado como este perrito

[Silvia♥ @silviia_08]

El resumen de mi vida universitaria