The Top Ten List of Sequencing Techniques

The journal Biotechniques has compiled  a list of the top ten sequencing techniques  complete with downloadable pdfs .

Prep of LB plates and LB media

LB media is the universal  growth media  in Molecular Biology. Although  LB  is often thought to  stand for  Luria Broth , in fact it was an abbreviation for lysogeny broth and LB should perhaps be more  correctly referred to as Luria Bertrani broth.  There are many variations,  but here  is our favorite  recipe:  

LB Agar plates
Dissolve  7.5 g of  Bacto Agar ( Difco ) and 12.5 g of   LB medium  ( MP Biochemicals)  in  water, Q to 500mL in a "Wheaton"  1 liter glass bottle.  Check that pH is 7, if not titrate with NaOH. Autoclave at 121 for 15 minutes and store at RT.  To pour plates, melt on hotplate and dispense.  If adding antibiotic , cool to 45 degrees centigrade, add the antibiotic and pour.

LB Media
Dissolve  12.5 g of   LB medium  ( MP Biochemicals)  in water, Q to 500mL in a "Wheaton"  1 liter glass bottle.  Check that pH is 7 , if not  titrate with NaOH.  Autoclave at 121 for 15 minutes and store at RT. 

3D print your favorite protein !

It is often difficult to appreciate the nuances of protein structure by viewing a spinning image on a monitor. Being able to look at a solid model,  often reveals major insights.  In this blog article , a simple step by step  protocol is presented that enables students  to create a 3D model of their favorite protein. All you need is a 3D printer !

Next generation DNA sequencing strategies

Dr. Mardis, one of the pioneers in using next generation sequencing strategies to  understand human disease, talks about recent developments.

The Million Mutation Project: A new approach to genetics in Caenorhabditis elegans

This is an interesting resource paper. The investigators have generated  a library  of  mutagenized    C. Elegans strains. What's new is that  each member of the library  has been sequence verified to identify  mutant alleles that cover all known C. Elegans genes.  The paper is  from the Waterston lab and is open access.

A Micro Benedict’s assay for the Determination of Glucose

The determination of glucose by Benedict's solution is a staple activity in Biotechnology  labs.  The classic format is to set up a large-scale reaction (5-10 ml) in glass tubes and boil until the desired reaction occurs. In the education lab this is a slow procedure and potentially fraught with problems.  Benedicts solution contains alkali and large volume boiling alkali solutions in an open tube are always a potential hazard.  Here, we suggest a micro procedure that can be done in a standard heating  block. 

Reactions are assembled in Eppendorf centrifuge tubes.  The unknown sample is Q’d to 250ul. Benedicts (250uL) is added and the tubes are closed (lid locks can be used if needed) and are heated at 70 degrees centigrade in a thermal block. Color development is monitored (usually takes 5-10 minutes) by negative and positive controls and the presence of glucose in the unknown determined.

In an inquiry driven exercise  this   rapid method is useful since it permits many determinations  of glucose in even a short lab session.

Here is an example from a recent  Inquiry exercise. 

 

Large Scale Restriction Enzyme Digestion of DNA

The  preparative digestion of DNA with restriction enzymes is a core technique in all Biotechnology labs. Here we describe a best practices approach.  One of the key factors in obtaining complete digestion of plasmid DNA is the  quality of the DNA.  Its important that  RF1 (or supercoiled ) plasmid is the predominant form. Presence of  substantial amounts of RFIII is usually indicative  of significant  DNA nicking.  Usually  the most important factor is that the DNA  is free from  the reagents used to purify it. For example,  even very small  traces of Phenol and  SDS can exert a significant inhibition of restriction enzyme activity.  Elsewhere is this blog,  we will describe a robust routine  method  of  plasmid DNA purification.

In the example below, 20ug of  PBR322 plasmid  were incubated  in a reaction (20uL)  containing  1X NEB buffer 2  and 10 units of HindIII. Before adding the enzyme,  a sample (1ul)  was withdrawn.  After incubation for 30 minutes at 37 degrees, another sample (1uL)  was withdrawn from the reaction.   The "before " and "after " samples were run  on 1% agarose gel electrophorsis.
As can be seen from  the picture,  complete digestion was obtained.  Subsequent work up of the DNA digest depends on its future utility  and will be discussed in that context in other posts.

 

The Bradford Reagent for Protein Determination

The Bradford Reagent  is the current  standard for protein determination, but it is quite expensive .   Most educational  exercises still use  the Biuret method since it is much more economical. However, the Bradford method is the one that  most  Biotechnology graduates  will  use in the workplace and so  it is important  to use this reagent in Biotechnology technique courses.

 Here, we describe  how to make  Bradford reagent  in economical fashion  from  readily available laboratory reagents.

Add Commassie brilliant blue ( 50mg) to  Methanol (50ml) , then  slowly add  100ml of phosporic acid , Q to 1 litre with dH20 .

TAE - A Classic Electrophoresis Buffer

TAE is available  commercially but can readily  be made in the Biotechnology  lab. Here is the recipe for a 50X stock.

To make 1 litre :
 Add  Tris base (242g) to  57 mL of Acetic Acid  and 100ml of 0.5M EDTA.  Q to 1 litre with dH2O .

Tips on PUBMED

In the two year college environment,  it can be difficult to access the full text  version of research papers.  Typically, after a PUBMED search ,  students can be discouraged when  they are faced  with a  journal paywall. However, unless the paper is very recent ( within the last 6 months)  the vast majority of papers  are available  through  PMC ( the free access version of PUBMED) . So  just "google"  NCBI   PMC  and that will take you to the front door !
For very recent papers , one can request a  pdf from the author.  In many journals , this is expedited by a simple online request.

An Economical Alternative to Ethidium Bromide for Visualization of DNA ?

Ethidium bromide is the classic reagent for the detection of nucleic acids on agarose gel electrophoresis. However, it is a recognized mutagen and care and attention are merited in its use. In the research lab, it is quite easy to regulate its use, since the personnel are aware of the  issues. However, in the education lab  the potential for accidental exposure is high.   Other less toxic reagents such as the SYBR series have  come into use, but they are significantly more expensive than Et BR.  So, in this post, we have examined whether the SYBR reagents can be directly added to the sample. If this works, it would avoid the need to include the visualization reagent in the agarose gel and thus be a significant saving, especially in an education environment. As shown in the picture,  increasing amounts of SYBR green  were added to 100ng of DNA marker ladders,  1ul of a 1/1000 dilution of SYBR green is sufficient to visualize 100ng of DNA (lane 3).

Thus SYBR  green  is a safe,  effective and economical reagent to visualize DNA.

Capital Loading Dye - Native Gel Electrophoresis

Sustainability is key to the running of Biotechnology courses. Paradoxically,  it's the small items that consume the most. One staple in the lab, is Native Gel loading dye which is used in  native gel electrophoresis  of DNA, ssRNA and Proteins.  Here is a reliable recipe  to make enough gel loading dye for a semester .

Fill up a  15ml  tube with  glycerol to  5ml , add  BPB (2ml of 1% ) , XC (2ml of 1% ) and EDTA ( 20uL  of 500mM) . Q with dH20  to 10 ml and aliquot  ( 100ul ). This provides a 10X loading dye.

The attached picture shows  plasmid DNA and ladder markers run  on a  1% agarose gel electrophoresis  in 1XTAE buffer using Capital Loading Dye. Image captured with iPhone .

A simple Semi-Quantitative Determination of Nucleic Acid

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Establishing a Biotechnology training program in a high school or two-year college environment is challenging.  Many of the basic techniques are quire sophisticated and use  “big ticket “ equipment items. In these posts we will explore ways to present Biotechnology using elements typically found in high school or two-year college labs.   One key problem with high school or two year environments is that   there are not working labs near by that can provide reagents that work, so troubleshooting is a significant endeavor.  Many Biotechnology methods take time to run, so it is an advantage to have a quick determination method. In this first example we are going to demonstrate a simple method to determine the presence of nucleic acids using SYBR  green, a UV light source and an iPhone or iPad.

Here is the protocol:

All volumes are in uL

Tube	tRNA (10mg/ml )	SYBR green 1/100	H2O (Q to)
1	-	1	100
2	1	1	100

tRNA is a convenient economical source of nucleic acid and has enough duplex structure. The reaction is assembled and placed on a UV transilluminator. A cardboard box with a hole serves as a portable darkroom and the image is recorded by an iPhone or iPad.