Category Archives: Lab Bench

Protocols, tips and advice for DNA, RNA and other molecular work in the lab.

Freeze and squeeze DNA extraction from gel

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Hi all,

Here’s a really easy DNA gel extraction:

1. Go about cutting out your bands of interest in the usual way.

2. Next cut the gel band(s) into tiny cubes.

3.  Place tiny cubes in an eppie.

4. Put eppie in -20oC for 15 to 30 min.  The idea is to make the band freeze into a mini ice cube.

5.  Knock out all of the gel band/cubes onto a 10 (long) x 5 (wide) cm piece of parafilm.

6. Fold the parafilm in half along the length while keeping your gel cubes along the fold in the parafilm.  You should have a piece of parafilm folded lengthwise with gel cubes sandwiched between two layers of plastic.

7.  Crush/squeeze the liquid out of the tiny cubes.  Liquid containing electrophoresis buffer and DNA will separate from the agarose.

8.  Collect DNA/electrophoresis buffer mixture with pipet and clean it by ethanol precipitation.

 

EDIT:  Borate in the electrophoresis buffer is a ligase inhibitor.  Make sure to thoroughly wash your DNA with 70% EtOH, at least 2 times.

Momma’s CTAB

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Like a few other people in the lab, I’ve been struggling with DNA extractions of late. I’ve tried several methods, including the “columnless” method that a few people are using. But I was having pretty spotty luck. Then, during a bout of methodological soul-searching, I also tried a CTAB protocol that used to be my go-to method. It failed me a few weeks ago, but in a second trial last week, it delivered the goods: loads of very pure, high molecular weight DNA. It might not work for all plants, and there do appear to be situations in which it is not the best option, but if anyone wants to get back to down-home, from-scratch extraction, the way momma used to do it, then give this one a try:

Please note: the results below are for Streptanthus (the plant I’m studying; Brassicaceae); I’m trying it for some Asteraceae right now, and will update the post once I have results.

Momma’s DNA Isolation via CTAB

Continue reading →

Home Made AMPure XP Beads

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AMPure XP beads are used to quickly clean PCR reactions and are also used in WGS library prep. You can also use them to do DNA size selections! They work well, but are very expensive. I found step-by-step instructions on how to make a home-made version online, purchased the reagents and tried the protocol. I tested the beads by attempting to clean up DNA ladder and I found that the home-made beads work identically to the AMPure XP beads.

Attached is the protocol for making the beads. I followed the protocol exactly. One note, I weighed the PEG-8000 in the fume hood because the fine powder is an irritant if inhaled. I also have attached a picture of my gel comparing clean-up of 100 bp DNA ladder with commercial AMPure XP beads and the home-made substitute.

Home-Made AMpure XP beads

AMPure clean-up comparison

Pipettes and Pipetting

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Hello All,

At the risk of insulting your intelligence I am posting some basic information about pipettes and pipetting so you won’t have any excuses for poor practice at the lab bench. The main reason that it is important to use our pipettes correctly is that we share them. Its not just your work that will be affected if you seriously contaminate a pipette or throw it out of calibration.

If you have an undergrad or new volunteer or employee doing anything with our pipettes show them this post and make them read the manual (below).

Continue reading →

Room Temperature RNA extraction

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Derivation: The following protocol is a modified Zhao Lai et al 2006 and TRizol Plus protocol.

Expected results: I tested the protocol with Arabidopsis seedlings and it gives quality RNA that was used for RT-PCR (non-quantitative).  I am able to achieve 75-270 ng/uL (100 uL elution – take this value with a grain of salt since it is derived from the nanodrop) of RNA from 38-48 mg of seedlings (this is approximately 20 seedlings grown for 18 days). I have yet to experience a failed RT-PCR with RNA isolated using this extraction.

Keys to success: Yield is highly linked to tissue mass and quality of tissue grind. The range of tissue mass is very small. One should use a small tissue amount and process a given sample over a column many times. Tissue is ground successfully only when it is not visible in the trizol/tissue/bead mixture. Minimize RNAse paranoia – Guanidine isothiocyanate does a good job of destroying protein structure, inactivating RNAses.

More after the jump Continue reading →

GBS protocol Version 2.0

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Hi all,

Here is the long awaited updated GBS protocol.

PROTOCOL ->>>> GBSv2.0

There are three main changes from the previous protocol.

-After digestion and ligation, all the product is kept so more attempts can be made at the PCR.

-The PCR uses Phusion Taq, has longer extension times and one additional cycle with more primers/Taq.

-Size selection is done using AMPure beads instead of gel extraction.

Continue reading →

GBS Protocol (GregO)

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Kristin and I have been working on GBS for a long time and since it now seems to be working, we wrote up a protocol. It is mostly the same from Greg Baute’s previous protocol, but with a few key changes (More DNA, more PCR). I’ve made it look nice and included a diagram for ease of thought.

Also, the official pronunciation of GBS is ‘jibs’

Continue reading →

Making Illumina Whole Genome Shotgun Sequencing Libraries – (Dan E.)

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I’ve been making whole genome shotgun sequencing libraries (for the purposes of this post: WGSS libraries) to sequence sunflower genomes on the Biodiversity Centre’s Illumina HiSeq. I haven’t been doing it for very long and its likely that my approach will change in the future as costs and products change but, as of early 2012, I’ve landed on a hybrid protocol based on kits from an outfit called Bioo Scientific. I use the Bioo Sci. adapter kit and their library prep kit up to the final PCR step at which point I switch to a PCR kit from another outfit called KAPA. I also use a KAPA kit to quant libraries with qPCR. In this post I give a little context then describe what I do to make WGSS libraries . . . Continue reading →

DNA ploidy and genome size estimation using flow cytometry (Dan B.)

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Flow cytometry (FCM) is – in principle – a relatively simple technique that allows one to measure properties of cells or organelles as they are intercepted (individually) by a high-intensity light source. In plant biology, it is often used to analyze extracts of intact nuclei, with the aim of determining DNA content and (indirectly) ploidy levels (this is the application I will focus on here). A good reference (including troubleshooting tips) can be found here.

Continue reading →

Illumina Sequencing Adapters and Barcodes (Dan E.)

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As of March 2012 we are using the Bioo Scientific NEXTflex barcoded adapters for WGS sequencing libraries made by ourselves, (well me so far). The set we are currently using comprises 48 barcodes, so we can multiplex up to a 48-plex in one lane on the Illumina HiSeq sequencer.

Bioo Sci. 48 barcoded adapters

Below are the sequences of the Illumina adapters and the 48 barcodes we are currently using. Continue reading →

Ampure XP beads – DNA clean up and size selection (Dan E.)

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Ampure XP magnetic beads have become popular for cleaning DNA from reactions and for size selecting DNA. WGS sequencing library preparation protocols, and the GBS protocol, involve one or more bead clean up steps. If you make next gen sequencing libraries you will use Ampure XP beads. Following is some basic information about them and my thoughts about using them, mainly for library preparations . . . Continue reading →

Loading Dye for Agarose Gels (Dan E.)

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If you are new to the lab bench, and perhaps even if you’re not, you may not know how to make loading dye for agarose gels. Its the sort of thing that tends to get made rarely and then persist, so people often inherit a supply and have no idea how to make more when they run out. For this sort of thing the best reference is the relevant appendix at the back of the Sambrook et al. Molecular Cloning text books (near Megan’s desk) and, of course, there is Google. Or, you can just do what I do. My recipe is incredibly simple and would be close to the cheapest option too. Continue reading →