gRNA design with efficient protospacer target cleavage

Different protospacers have different efficiency for producing mutations. In other words not every spacer in front of the PAM (protospacer adjacent motif) will be cut with the same efficiency as every other spacer. Software like CHOPCHOP can help you to design gene editing experiments with a much high rate of success.

gRNA design video without intro
gRNA design video with intro

Important considerations

  • You want your gRNA to cut your target with high efficiency but not to cut other parts of the genome. Software helps for this.
  • Mismatches in the seed region of the target sequence are more likely to prevent your gRNA from cutting than mismatches further from the PAM.

Designing and making CRISPR/Cas9 plasmids-quick & easy

Cloning work for making plasmids can be slow and frustrating. However, making plasmids (constructs) for gene editing can be a piece of cake. Thanks to modern techniques, pipetting is pretty much the only skill you need to make CRISPR constructs to knockout genes. Here is an Excel file with a protocol for cloning gRNA protospacers. No gel purification is necessary. Efficiency is about 100%.

gRNA spacer cloning video with intro
gRNA spacer cloning video without intro

You will want to sequence your final CRISPR constructs. On rare occasion I have found a base pair error in the protospacer sequence. This can occur if the oligonucleotides have a small portion that are missing bases from what you have ordered. The purity of oligonucleotides is not always absolutely perfect from some suppliers. Sequencing two clones will virtually guarantee you get the final construct you want.

Plasmid DNA Isolation – Improved Yield, No Kit

This improved plasmid DNA isolation has greater yield than most any column based method and costs about 5 cents. A number of steps have been optimized to produce large quantities of ultra pure DNA. Yield can exceed 40 micrograms plasmid per 1.7 mL culture. The low cost makes this procedure friendly to any lab on a budget or labs that wish to use money on more pressing commodities, like data. A longer version of this video with more tips can be found here.

Short version of Plasmid DNA Isolation (Recommended).

Download a print Plasmid DNA Isolation Protocol here.

Extracción de ADN plasmídico-Alto rendimiento, económico

Procedimiento de extracción de ADN plasmídico de alto rendimiento y económico, basado en un método mejorado de lisis alcalina. Este protocolo puede producir 43 microgramos de ADN de alta calidad a partir de 1.7 mL de cultivo bacteriano. Además es más rápido y cuesta solo 5 centavos. No requiere kits caros, lo que es perfecto para laboratorios con poco presupuesto.

Puedes descargar el protocolo aquí Extracción-de-ADN-plasmídico.

Tobacco Agroinfiltration – Transient Transformation

Transient Transformation of Wild Tobacco by Agroinfiltration

Transiently transforming Nicotiana benthamiana (wild tobacco) by agroinfiltration is an easy way to quickly express genes for protein localization or gene function studies. This video shows you how to perform tobacco agroinfiltration.

Download a written protocol for Tobacco Agroinfiltration here.

Gel purification – Freeze & Squeeze method

Very fast gel purification by freeze & squeeze

Gel purification by the Freeze & Squeeze method is probably the fastest way to get DNA out of a gel. This gel purification method is also the least labor intensive method. The video below shows you how to make filters for Freeze & Squeeze gel extraction and how to perform the extraction

Advantages of the Freeze & Squeeze gel purification method

  1. Very fast (just over 8 minutes)
  2. Minimally labor intensive
  3. Very cheap (about 4 cents)

Download a print protocol for the Freeze and Squeeze Gel Extraction here.

DNA extraction – improved phenol:chloroform method

DNA extraction (PCI) of PCR or other enzyme reactions

Phenol:Chloroform:Isoamyl alcohol (25:24:1) (PCI) DNA extraction can be used to clean up DNA from pretty much any sample. After trying lots of methods like kits with columns, I prefer the PCI method over any other method for applications like PCR purification. This PCI method gives better yield than column based methods and the DNA is suitable for any application like sequencing, ligating, or Gibson Assembly. The video below describes an improved PCI extraction that gives the best yield and purity, is very fast, and costs next to nothing.

Advantages of this Phenol:Chloroform DNA Extraction

  • Results in DNA of high yield and purity.
  • Fast
  • Cheap (costs just a few cents including tubes)
  • Eco-friendly. Produces much less plastic waste than column based methods. Produces 10 times less phenol:chloroform waste than traditional PCI methods.
  • Extremely scalable. Works for any range between femtograms and milligrams of DNA.

Down load a printable phenol:chloroform DNA extraction protocol here.

Using a micropipette

Correctly using a micropipette

Molecular biology requires the manipulation of micro-liter quantities of liquid. Thus the micropipette is one of the most frequently used pieces of equipment in the lab. This video will help get newbie’s skills with micropipettes up to speed quickly.

The video is for using Eppendorf brand micropipettes. Gilson pipets can be used in a near identical manner and this video can help you to set the pickup volume with Gilson micropipettes.

Statistical analysis, t-test in Excel

Statistical analysis

Statistics are one of the most important tools for a scientist. They allow scientists to test hypotheses. Perhaps you would like to  know if a treatment makes your favorite organism grow better. Statistical analysis would let you determine the probability that the treatment increases growth. The below video shows you how to perform statistical analysis (t-test) in Excel.

Choice of t-test (paired vs two sample equal variance vs two sample unequal variance)

Ideally statistical analysis should be planned before the experiment is setup. Paired t-test has more statistical power than the other two types of t-test because it helps minimize the effect of nuisance factors that confound the experiment results. An example of how to setup an experiment to take advantage of paired t-test in plants is as follows. Control and treatment for each replicate would be planted side by side so that the replicate was in a block. Doing this minimizes the effect that different growing positions have on each replicate. A paired t-test takes this into consideration and assumes while the absolute measurement values might change for different replicates, the mean difference between control and treatment for each replicate should be conserved. The best part of using a paired t-test or other blocking strategy, is that replicates do not need to be completed at the same time. This way there is no real limit to the number of replicates for an experiment.

Quantifying SDS-PAGE bands

Quantifying bands on SDS-PAGE using ImageJ

The below video shows you how to use ImageJ to quantify bands on a SDS-PAGE gel image. In this example, bands are normalized to the total protein in the lane.

Here you can download the sample gel image from the video to practice with. Note that many commercial gel docs, like Bio-rad, come with software for quantifying SDS-PAGE bands. In contrast, ImageJ is a free alternative that can work with any gel images including ones generated with an office scanner. Almost the same procedure in the video can be used for quantifying nucleic acid gels or even features on non-gel images.