Hi all,
I’m not sure how most of you were sharing your gels but I set up a dropbox folder within the Rieseberg gels folder. Sending e-mails was tiresome. If you’re interested in synchronizing your gels with your computer drag your folder(s) to the dropbox folder. The dropbox account credentials (online and client) are as follows:
user=allandebono@allandebono.org
password=1d7hz@!
… Go through the process as usual to set up a shared folder.
Cutting a slice from an agarose gel in order to size select DNA or to isolate a PCR product etc. is standard practice in lab genetics. This post is about how I do it in our lab, (as of Feb 2012).
There are three features of my current method that may be unfamiliar to you even if you’ve done this sort of thing before:
Here is the WGA Qiagen REPLIg ultra fast PROTOCOL.
I added also general information (mostly from the Qiagen site) about the REPLI-g method of amplification and about other similar Qiagen kits available.
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We have some columns in the lab that are for cleaning and concentrating genomic DNA. They are made by an outfit called Zymo Research and should be easy to find, look for a bright yellow box.
This post describes a test I ran of these columns. Continue reading
This post is about fragmenting, or shearing, genomic DNA to a particular size range using the Rieseberg lab’s Bioruptor sonicator.
Most of the current whole genome shotgun (WGS) library preparation protocols for NGS applications start with fragmented DNA. Generally speaking, this starting DNA should be a certain size and, for multiple samples, consistently that size. This objective turns out to be quite a tricky thing to accomplish with the Bioruptor. Given that WGS sequencing will probably continue to be popular in the lab, I am posting here what I have learned so far about taking whole genomic sunflower DNA and smashing it to the size range that I want using the Bioruptor. If I discover anything else in future library preps I’ll add it below. If anybody else has useful tips please comment.
This is a protocol for amplifying very large amplicons that are high in AT. I developed in order to amplify the 8.2 Kb region containing the promoter of Arabidopsis FT which is approximately 70% AT.
In my hands it worked reliably with an 8 Kb amplicon (DeBono notebook 1, July 2010) but can be easily modified for longer products using more dNTP and optimized template concentration.
Running a RNA Nano chip is very similar to running a DNA chip on the Bioanalyzer. However there are some things to keep in mind:
1. Clean everything with RNAse Away! before you start. I clean the chip loading stand as well as everything I am working with.
2. Make sure the reagents are at room temperature before you start to make the gel.
3. Before loading the chip, pipette 1.2 uL of each sample as well as the ladder into PCR tubes and incubate in the thermocycler for 2 minutes.
On the last point, I aliquot my samples into the PCR tubes and incubate them right before I load the gel. The instructions say to denature all the ladder in one go, but I have had better results when I denature an aliquot with my samples right before I run the chip.
Attached is a chip of 12 conifer leaf tissue extractions that worked out pretty well.
The bioanalyzer can be useful for quantifying and quality checking double stranded DNA, RNA, and proteins. This method of gel electrophoresis will tell you both fragment size and concentration for each fragment, using up only 1 uL of sample. The reagents and chips are expensive (for the High Sensivity chips, ~$100/11 samples/single use chip), and have limited shelf life (4 months or less).
Rieseberg lab members have used this machine to check the quality of sequencing libraries and cDNA for microarray expression analyses. Various DNA and RNA kits are available, depending on what you want to measure, and their specs are at the bottom of this post.
Here are some of the lab’s basic protocols for:
Column based DNA extraction version 2
This post is about quantifying DNA samples in the Rieseberg lab, i.e. using the tools we have available to us in our lab in the Biodiversity building as of November 2011.
There are three ways to quantify the amount of DNA in an aqueous solution (e.g. DNA dissolved in water or TE):
These methods have different benefits and limitations but they are all valid and useful.
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This is a protocol for extracting good yields of high quality DNA from sunflower leaf tissue.
The appealing thing about this protocol is the high yield of high quality DNA that it can produce from an easily achievable amount of starting material. The downside is that it is labour intensive and involves dangerous chemicals. See below for elaboration.
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