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Saturday, 4 February 2012

How to Dilute a Stock Solution

How to Dilute a Stock Solution

A. Why dilute? 

Here are two situations that arise repeatedly in molecular biology labs:
1. You have a stock solution of some compound,  let’s say an antibiotic, and you want to add the
compound to growth medium, at a much smaller concentration than the stock solution.
2. You have a tube of very concentrated bacteria, perhaps a billion cells per milliliter. You want to put
a few hundred of them on a petri plate, so that the colonies that arise will be easily distinguishable.
In both cases, the way out of the problem is to dilute the original solution. If you work in a lab, you need to
know how to do this.

B. Methods of calculating dilutions 

1. DILUTION FACTOR METHOD (fast, but requires inspiration): First, figure out the factor by which the
original solution must be diluted. Second, divide the final volume of the desired solution by that
factor, yielding the volume required of the original solution.
EXAMPLE: Suppose you need to make a 3 ml solution of growth medium supplemented with 50 µM of
the antibiotic ampicillin from a stock solution of 5 mM ampicillin. The dilution factor is :
  (5 mM) / (50 µM) = (5000 µM) / 50 µM) = 100
 so you need to dilute:
   (3 ml) / 100 = (3000 µl) / 100 = 30 µl
 of the stock solution to a final volume of 3 ml.

Sunday, 25 December 2011

Isolation of Casein from Milk

  Isolation and Identification of Casein From Milk Course Notes

Milk is the probably the most nutritionally complete food found in nature. Whole milk contains vitamins (principally thiamine,riboflavin, panthothenic acid and vitamins A, B12 and D), minerals (calcium, sodium, phosphorus, potassium, and trace minerals), proteins (which include all the essential amino acids), carbohydrates (mostly lactose), and lipids (fats). Whole milk is an oil in water emulsion, containing approximately 4% fat dispersed as very small (micron sized) globules. The fat emulsion is stabilized by complex phospholipids and proteins that are absorbed on the surface of the emulsion. Since the fat in milk is so finely dispersed it is more easily digested than fats from any other source.

Isolation of Casein, Lactose, and Albumin from Milk

Milk is a food of exceptional interest. Not only is milk an excellent food for the very young, but humans 
have also adapted milk, specifically cow’s milk, as a food substance for persons of all ages. Many 

specialized milk products like cheese, yogurt, butter, and ice cream are staples of our diet.

Experiment 11: Isolation and Characterization of Casein from Milk

Adapted from Experiment 21, “Isolation of Protein, Carbohydrate and Fat from Milk”, in
Mohr. S.C., Griffin, S.F., and Gensler, W. J. Laboratory Manual for Fundamentals of
Organic and Biological Chemistry by John McMurry and Mary E. Castellion,: nglewood
Cliffs, Prentice-Hall, 1994 and Wayne P. Anderson (4/2002)

Purpose: In this lab you will be isolating the proteins casein and lactalbumin from a sample of milk. You
will use these values to determine the percent protein in milk compared to the listed value on the box.


Saturday, 24 December 2011

Polony PCR Amplification

This section outlines the protocol for single-molecule amplification within the acrylamide gel.  The protocol given here may differ in specifics from what is presented here, but reflects what we are doing currently (Summer 2003).  We have tried to include excessive detail here but let us know if anything is unclear.  The general steps are:

1.    Cast acrylamide gels
2.    Diffuse in PCR reagents
3.    PCR amplification
4.    Slide clean-up

Further Protocol 

Long PCR Protocol Reagents and Guidelines

General Guidelines for Long PCR Conditions and Enzyme Mixtures
Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Following the results of Cheng et al.
(1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases have been used successfully for many applications. The buffer listed below works well with Tth and Vent, but not with others. If you are interested in using other polymerases make sure that you use compatible buffers. Error rates for other polymerases can be found at 

Read Full Protocol Here

Qiagen RNeasy Plant RNA Isolation

RNA isolation
RNeasy Kits are designed to isolate total RNA from small quantities of starting material. They provide a fast and simple method for the preparation of up to 100 µg total RNA from animal cells and tissues, bacteria, and yeast (RNeasy Mini Kits) or plant cells and tissues and filamentous fungi (RNeasy Plant Mini Kits). RNeasy Plant Mini Kits are fast and avoid tedious methods, such as CsCl step-gradient ultracentrifugation and alcohol precipitation steps, or methods involving the use of toxic substances such as phenol and/or chloroform. The purified RNA is ready for use in standard downstream applications such as RT-PCR, Northern, dot, and slot blotting, poly A+ RNA selection, primer extension, RNase and S1 nuclease protection, cDNA synthesis, differential display, expression-array and expression-chip analysis.

Formaldehyde Agarose Gel Electrophoresis Protocol

The following protocol for formaldehyde-agarose gel electrophoresis gives enhanced sensitivity for gel and subsequent analysis (e.g. northern blotting). A key feature is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols (e.g. Sambrook, J. et al., eds. (1989) Molecular cloning — a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

1.2% Formaldehyde Agarose gel preparation
To prepare Formaldehyde Agarose gel (1.2% agarose) of size 10 x 14 x 0.7 cm, mix:
1.2 g agarose
10 ml 10x Formaldehyde Agarose gel buffer (see composition below)
Add RNase-free water to 100 ml
If smaller or larger gels are needed, adjust the quantities of components proportionately. Heat the mixture to melt agarose. Cool to 65°C in a water bath. Add 1.8 ml of 37% (12.3 M) formaldehyde (toxic) and 1 µl of a 10 mg/ml ethidium bromide (mutagenic) stock solution. Mix thoroughly and pour onto gel support. Prior to running the gel, equilibrate in 1x Formaldehyde Agarose gel running buffer for at least 30 min.

RNA sample preparation for Formaldehyde Agarose gel electrophoresis
Add 1 volume of 5x loading buffer per 4 volumes of RNA sample (for example 10 µl of loading buffer and 40 µl of RNA) and mix.
Incubate for 3–5 min at 65°C, chill on ice, and load onto the equilibrated Formaldehyde Agarose gel.

Gel running conditions
Run gel at 5–7 V/cm in 1x Formaldehyde Agarose gel running buffer.

Composition of Formaldehyde Agarose gel buffers

10x Formaldehyde Agarose Gel buffer
200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)
50 mM sodium acetate
10 mM EDTA
pH to 7.0 with NaOH

1x Formaldehyde Agarose Gel Running Buffer
100 ml 10x Formaldehyde Agarose gel buffer
20 ml 37% (12.3 M) formaldehyde
880 ml RNase-free water

5x RNA Loading Buffer
16 µl saturated aqueous bromophenol blue solution†
80 µl 500 mM EDTA, pH 8.0
720 µl 37% (12.3 M) formaldehyde
2 ml 100% glycerol
3084 µl formamide
4 ml 10 x Formaldehyde Agarose gel buffer
RNase-free water to 10 ml
Stability: Approximately 3 months at 4°C

2M H2SO4 for 100ml

2M H2SO4 for 100ml
  • mix 50ml Milli Q water + 50ml 4M H2SO4 solution

Science Protocols