tag:blogger.com,1999:blog-9842222753472606702024-02-07T17:35:26.051-08:00Oxford Laboratory TechnologyLatest Laboratory Related Science and Technology: Find New E-books, Protocols, Softwares, Animations, News and Much more..Unknownnoreply@blogger.comBlogger391125tag:blogger.com,1999:blog-984222275347260670.post-20087382181878757402014-11-09T00:12:00.001-08:002014-11-09T00:12:23.132-08:00TE buffer (Tris-EDTA buffer) Recipe<div dir="ltr" style="text-align: left;" trbidi="on">
The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.<br />
<br />
10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA<br />
<br />
A stock can be prepared and stored at RT<br />
<br />
Component For 500ml<br />
1M Tris pH 8* 5ml<br />
0.5M EDTA pH 8 1ml<br />
dH2O 496ml<br />
<br />
Autoclave to sterilize. It is then best to remove a working aliquot and not repeatedly access the stock.<br />
*the pH is usually adjusted to RNA 8.0 for DNA and 7.5 for</div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-21411258462937995612014-11-09T00:10:00.001-08:002014-11-09T00:10:08.342-08:00How to make acetate-acetic acid buffer solutions<div dir="ltr" style="text-align: left;" trbidi="on">
Sodium acetate-acetic acid buffer solutions, pH 3.7-5.6<br />
<br />
Sodium acetate trihydrate, CH3COONa.3H2O, M.Wt. 136.09;<br />
0.2M solution contains 27.22g/l.<br />
Acetic acid, glacial is ~17.47M.<br />
<br />
x ml 0.2M NaOAc and y ml 0.2M HOAc mixed.<br />
<br />
pH, 18°C X mL<br />
<br />
0.2M NaOAc<br />
<br />
Y mL<br />
<br />
0.2M HOAc<br />
3.7 10.0 90.0<br />
3.8 12.0 88.0<br />
4.0 18.0 82.0<br />
4.2 26.5 73.5<br />
4.4 37.0 63.0<br />
4.6 49.0 51.0<br />
4.8 59.0 41.0<br />
5.0 70.0 30.0<br />
5.2 79.0 21.0<br />
5.4 86.0 14.0<br />
5.6 91.0 9.0<br />
<br />
<br /></div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-10196031665634394122014-05-11T17:00:00.001-07:002014-05-11T17:00:02.862-07:00How to make 1x buffer from 20x<div dir="ltr" style="text-align: left;" trbidi="on">
As explained before <a href="http://labclone.blogspot.co.uk/2014/05/how-to-make-1x-buffer-from-10x.html" target="_blank">here </a>it is very simple and you need to understand the concept.<br />
or you can use the formula M1V1=M2V2<br />
<br />
Example<br />
<br />
You have 20x Orange Juice and you wanna make 10 ml of 1x.<br />
<br />
Simple use M1V1=M2V2<br />
<br />
M1= Original Concentration [which is 20x]<br />
V1= required volume to make 1x<br />
<br />
M2= Desired Concentration [which is 1x]<br />
V2= Desired volume which is 10 ml<br />
<div>
<br /></div>
<div>
20x * V1 = 1x * 10ml</div>
<div>
<br /></div>
<div>
V1= 0.5 ml of 20x concentrated juice is needed to add in 9.5 ml of water [10-0.5=9.5 ml]</div>
<div>
<br /></div>
<div>
IMPORTANT </div>
<div>
<br /></div>
<div>
Don't add 0.5 ml in 10 ml. This will make final volume 10.5 ml and your concentration will be wrong. </div>
<div>
<br /></div>
<div>
<br /></div>
<div>
<br /></div>
<div>
<br /></div>
<br />
<br /></div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-75215262744997312572014-05-11T16:47:00.002-07:002014-05-11T16:47:31.160-07:00How to make 1x BSA<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
How to make 1x bsa or Bovine serum albumin (BSA)<br />
<br />
1x BSA generally suggest that you want make 1 gram per litre BSA solution.<br />
<br />
You can use the following method to make<br />
Material<br />
<br />
Phosphate buffered<br />
<br />
saline (PBS), pH 7.4, 10X None PS00000041 10X 50 ml 1X<br />
<div>
Sigma-Aldrich A4919 Powder 0.5 g 1 g/L </div>
<br />
Preparation:<br />
<br />
1. Weigh 0.5 g BSA and add to 300 ml purified water in a 600-ml beaker.<br />
2. Add 50 ml of 10X PBS.<br />
3. Adjust volume to 500 ml.<br />
4. Store at room temperature in an appropriately labeled glass bottle.<br />
<div>
<br /></div>
</div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-76121061361704342702014-05-11T16:41:00.002-07:002014-05-11T16:41:28.504-07:00How to make 1x buffer from 10x<div dir="ltr" style="text-align: left;" trbidi="on">
This is very simple<br />
<br />
You just need to learn the concept.<br />
<br />
10x means 10 times concentrated<br />
<br />
you need to make 1x<br />
<br />
so you need to dilute it 10 time.<br />
<br />
of you can take 1 part of 10x and mix with 9 part of water [1+9=10] to make 10x buffer.<br />
<br />
Or you can use formula below make 1x buffer from 10x concentration buffer<br />
<br />
M1V1=M2V2<br />
<br />
M1 = stock concentration [10x]<br />
V1 = volume needed of M1 concentrated stock [Z]<br />
<br />
M2 = desired concentration [1x]<br />
V2 = desired volume [say 10 ml you wanna make]<br />
<br />
So as per M1V1=M2V2<br />
<br />
10x * Zml = 1x *10ml<br />
<br />
Z =1ml of 10x you need in 10ml of water.<br />
<br />
This also means you need to add 10-1=9 ml of water in 1 ml of 10x concentrate to make 1x buffer.<br />
<br />
<b>IMPORTANT </b><br />
<br />
Don't add 1ml in to 10 ml. which would be 11 ml and your concentration will be wrong.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br /></div>
Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-56086063475287358482012-02-04T08:09:00.000-08:002012-02-04T08:09:04.834-08:00How to Dilute a Stock Solution<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<b>How to Dilute a Stock Solution</b><br />
<b><br /></b><br />
<b>A. Why dilute? </b><br />
<br />
Here are two situations that arise repeatedly in molecular biology labs:<br />
1. You have a stock solution of some compound, let’s say an antibiotic, and you want to add the<br />
compound to growth medium, at a much smaller concentration than the stock solution.<br />
2. You have a tube of very concentrated bacteria, perhaps a billion cells per milliliter. You want to put<br />
a few hundred of them on a petri plate, so that the colonies that arise will be easily distinguishable.<br />
In both cases, the way out of the problem is to dilute the original solution. If you work in a lab, you need to<br />
know how to do this.<br />
<br />
<b>B. Methods of calculating dilutions </b><br />
<br />
1. DILUTION FACTOR METHOD (fast, but requires inspiration): First, figure out the factor by which the<br />
original solution must be diluted. Second, divide the final volume of the desired solution by that<br />
factor, yielding the volume required of the original solution. <br />
EXAMPLE: Suppose you need to make a 3 ml solution of growth medium supplemented with 50 µM of<br />
the antibiotic ampicillin from a stock solution of 5 mM ampicillin. The dilution factor is :<br />
(5 mM) / (50 µM) = (5000 µM) / 50 µM) = 100<br />
so you need to dilute:<br />
(3 ml) / 100 = (3000 µl) / 100 = 30 µl <br />
of the stock solution to a final volume of 3 ml.<br />
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-72733323291595804552011-12-25T03:04:00.001-08:002011-12-25T03:05:40.011-08:00Isolation of Casein from Milk<div dir="ltr" style="text-align: left;" trbidi="on">
<span style="font-size: x-small;"><br /></span><br />
<h1 style="text-align: left;">
<span style="font-size: x-small; font-weight: normal;"> Isolation and Identification of Casein From Milk Course Notes</span></h1>
<h1 style="text-align: center;">
<span style="font-size: x-small; font-weight: normal;"><span style="text-align: left;">Milk is the probably the most nutritionally complete food found in
nature. Whole milk contains vitamins (principally thiamine,riboflavin,
panthothenic acid and vitamins A, B</span><sub style="text-align: left;">12</sub><span style="text-align: left;"> 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.</span></span></h1>
<div>
<span style="font-size: x-small;"><span style="text-align: left;"><a href="http://homepages.ius.edu/dspurloc/c122/casein.htm" target="_blank">Read Full Protocol Here</a></span></span></div>
<div>
<span style="font-size: x-small;"><br /></span></div>
<div>
<span style="font-size: x-small;">Isolation of Casein, Lactose, and Albumin from Milk</span></div>
<div>
<span style="font-size: x-small;"><br /></span></div>
<div>
<span style="font-size: x-small;"></span><br />
<div>
<span style="font-size: x-small;">Milk is a food of exceptional interest. Not only is milk an excellent food for the very young, but humans </span><br />
<div style="display: inline !important;">
<span style="font-size: x-small;">have also adapted milk, specifically cow’s milk, as a food substance for persons of all ages. Many </span></div>
<span style="font-size: x-small;">
</span><br />
<div style="display: inline !important;">
<span style="font-size: x-small;">specialized milk products like cheese, yogurt, butter, and ice cream are staples of our diet.</span></div>
<span style="font-size: x-small;">
</span></div>
<span style="font-size: x-small;">
</span><br />
<div>
<span style="font-size: x-small;"></span><br />
<div style="display: inline !important;">
<span style="font-size: x-small;"><br /></span></div>
<span style="font-size: x-small;">
</span></div>
<span style="font-size: x-small;">
<div>
<br />
<div style="display: inline !important;">
<a href="http://courses.chem.psu.edu/chem36/Web%20Syn06/Exp112Syn06.pdf" target="_blank">Read Full Protocol Here.</a></div>
</div>
<div>
<br /></div>
<div>
Experiment 11: Isolation and Characterization of Casein from Milk</div>
<div>
<br /></div>
<div>
<div>
Adapted from Experiment 21, “Isolation of Protein, Carbohydrate and Fat from Milk”, in</div>
<div>
Mohr. S.C., Griffin, S.F., and Gensler, W. J. Laboratory Manual for Fundamentals of</div>
<div>
Organic and Biological Chemistry by John McMurry and Mary E. Castellion,: nglewood</div>
<div>
Cliffs, Prentice-Hall, 1994 and Wayne P. Anderson (4/2002)</div>
<div>
<br /></div>
<div>
<a href="http://facstaff.bloomu.edu/mpugh/Experiment%2011.pdf" target="_blank">Read Full Experimental Protocol Here</a></div>
<div>
<br /></div>
<div>
<div>
ISOLATION OF CASEIN FROM MILK</div>
<div>
Purpose: In this lab you will be isolating the proteins casein and lactalbumin from a sample of milk. You</div>
<div>
will use these values to determine the percent protein in milk compared to the listed value on the box.</div>
</div>
</div>
<div>
<br /></div>
<div>
<a href="http://www.apsu.edu/sites/apsu.edu/files/chemistry/SP11_1021_ISOLATION_OF_PROTEINS_FROM_MILK.pdf" target="_blank">Read Full Experimental Protocol here.</a></div>
</span></div>
<h1 style="text-align: left;">
<span style="font-size: x-small; font-weight: normal;"> </span></h1>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-59232923645548476452011-12-24T17:24:00.001-08:002011-12-24T17:25:05.138-08:00Polony PCR Amplification<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<span style="font-family: Arial; font-size: x-small;">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:</span><br />
<br />
<span style="font-family: Arial; font-size: x-small;">1. Cast acrylamide gels</span><br />
<span style="font-family: Arial; font-size: x-small;">2. Diffuse in PCR reagents</span><br />
<span style="font-family: Arial; font-size: x-small;">3. PCR amplification</span><br />
<span style="font-family: Arial; font-size: x-small;">4. Slide clean-up</span><br />
<span style="font-family: Arial; font-size: x-small;"><br /></span><br />
<span style="font-family: Arial; font-size: x-small;"><a href="http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm" target="_blank">Further Protocol</a> </span><br />
<span style="font-family: Arial; font-size: x-small;"><br /></span><br />
<span style="font-family: Arial; font-size: x-small;"><br /></span></div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-31939868575580614432011-12-24T17:22:00.001-08:002011-12-24T17:22:52.239-08:00Long PCR Protocol Reagents and Guidelines<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<h2>
</h2>
<h2>
</h2>
<h2>
</h2>
<h2>
<span style="font-size: x-small; font-weight: normal;">General Guidelines for Long PCR Conditions and Enzyme Mixtures</span><span style="font-size: x-small; font-weight: normal;"><br />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. </span><a href="http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html#Cheng" style="font-size: small; font-weight: normal;">(1)</a><span style="font-size: x-small; font-weight: normal;"> we have had success using </span><span style="font-size: x-small; font-weight: normal;">Tth</span><span style="font-size: x-small; font-weight: normal;"> (ABI/Perkin-Elmer) as the main-component polymerase and </span><span style="font-size: x-small; font-weight: normal;">Vent</span><span style="font-size: x-small; font-weight: normal;">
(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 </span><a href="http://research.nwfsc.noaa.gov/protocols/taq-errors.html" style="font-size: small; font-weight: normal;">http://research.nwfsc.noaa.gov/protocols/taq-errors.html </a></h2>
<a href="http://arep.med.harvard.edu/labgc/estep/longPCR_protocol.html" target="_blank"><span style="font-size: x-small; font-weight: normal;">Read Full Protocol Here</span></a><br />
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-51047854621582183392011-12-24T17:16:00.002-08:002011-12-24T17:16:54.327-08:00Qiagen RNeasy Plant RNA Isolation<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">RNA
isolation<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">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 (</span><b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">RNeasy Mini
Kits</span></b><span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">)
or plant cells and tissues and filamentous fungi (</span><b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">RNeasy Plant
Mini Kits</span></b><span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">). 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.<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;"><o:p><a href="http://abe.leeward.hawaii.edu/Protocols/RNeasy%20Principle.htm" target="_blank">Further Protocol Information</a></o:p></span></div>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-61083809998025221042011-12-24T17:16:00.000-08:002011-12-24T17:16:01.599-08:00Formaldehyde Agarose Gel Electrophoresis Protocol<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<div class="Section1">
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">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) </span><i><span style="font-family: Futura-BookOblique; mso-bidi-font-family: Futura-BookOblique;">Molecular cloning — a laboratory
manual, </span></i><span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press).<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">1.2% Formaldehyde
Agarose gel preparation<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">To prepare Formaldehyde
Agarose gel (1.2% agarose) of size 10 x 14 x 0.7 cm, mix:<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">1.2 g agarose<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">10 ml 10x Formaldehyde
Agarose gel buffer (see composition below)<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">Add RNase-free
water to 100 ml<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">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 (t</span><i><span style="font-family: Futura-BookOblique; mso-bidi-font-family: Futura-BookOblique;">oxic</span></i><span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">) and 1 µl of a 10 mg/ml ethidium
bromide (<i>mutagenic</i>) 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.<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">RNA sample
preparation for Formaldehyde Agarose gel electrophoresis<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">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.<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">Incubate for
3–5 min at 65°C, chill on ice, and load onto the equilibrated Formaldehyde
Agarose gel.<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">Gel running
conditions<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">Run gel at 5–7
V/cm in 1x Formaldehyde Agarose gel running buffer.<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">Composition
of Formaldehyde Agarose gel buffers<o:p></o:p></span></b></div>
</div>
<b><span style="font-family: Futura-Heavy; font-size: 11.0pt; mso-ansi-language: EN-US; mso-bidi-font-family: Futura-Heavy; mso-bidi-language: AR-SA; mso-fareast-font-family: "MS Mincho"; mso-fareast-language: JA;"><br clear="all" style="mso-break-type: section-break; page-break-before: auto;" />
</span></b>
<div class="Section2">
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">10x Formaldehyde
Agarose Gel buffer<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">200 mM
3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">50 mM sodium
acetate<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">10 mM EDTA<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">pH to 7.0 with
NaOH<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">1x Formaldehyde
Agarose Gel Running Buffer<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">100 ml 10x Formaldehyde
Agarose gel buffer<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">20 ml 37%
(12.3 M) formaldehyde<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">880 ml
RNase-free water<o:p></o:p></span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<b><span style="font-family: Futura-Heavy; mso-bidi-font-family: Futura-Heavy;">5x RNA
Loading Buffer<o:p></o:p></span></b></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">16 µl
saturated aqueous bromophenol blue solution†<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">80 µl 500 mM
EDTA, pH 8.0<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">720 µl 37%
(12.3 M) formaldehyde<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">2 ml 100%
glycerol<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">3084 µl
formamide<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">4 ml 10 x Formaldehyde
Agarose gel buffer<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">RNase-free
water to 10 ml<o:p></o:p></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;">Stability:
Approximately 3 months at 4°C</span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;"><br /></span></div>
<div class="MsoNormal">
<span style="font-family: Futura-Book; mso-bidi-font-family: Futura-Book;"><a href="http://abe.leeward.hawaii.edu/Protocols/Formaldehyde%20Agarose%20Gel%20for%20RNA.htm" target="_blank">Ref</a></span></div>
</div>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-71268835506385454002011-12-24T17:12:00.003-08:002011-12-24T17:14:24.579-08:002M H2SO4 for 100ml<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<span class="szoveg2">2M H<sub>2</sub>SO<sub>4</sub> for 100ml</span><span class="szoveg3"><cite></cite></span><br />
<ul>
<li>mix 50ml Milli Q water + 50ml 4M H<sub>2</sub>SO<sub>4</sub> solution</li>
</ul>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-82312415167507379822011-12-24T17:12:00.001-08:002011-12-24T17:14:24.589-08:004M H2SO4 for 1 liter<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<span class="szoveg2">4M H<sub>2</sub>SO<sub>4</sub> for 1 liter</span><span class="szoveg3"><cite></cite></span><br />
<ul>
<li>Carefully pour 392ml H<sub>2</sub>SO<sub>4</sub> {Sulphuric acid 96% H<sub>2</sub>SO<sub>4</sub> Carlo Erba reagents Code no. 410301 (Reanal 17769) FW 98.078} into 608ml Milli Q water (always pour acide into water)</li>
</ul>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-10722574298225285222011-12-24T17:11:00.005-08:002011-12-24T17:14:24.533-08:00TMB-buffer for 1 liter<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<span class="szoveg2">TMB-buffer for 1 liter</span><span class="szoveg3"><cite>[0.1M Na-acetate, pH 5.5]</cite></span><br />
<ul>
<li>Disolve in 950ml Milli Q water </li>
<ul>
<li>13.6g Na-acetate {Nátrium acetát, kristályvizes CH<sub>3</sub>COONa*3H<sub>2</sub>O M:136.08 Reanal 14021}</li>
</ul>
<li>Adjust pH to 5.5 with app. 700ul cc. Acetic acid {Ecetsav 96% Reanal 0914-1-08-65 M:60.05}</li>
<li>Adjust volume to 1 liter with Milli Q water </li>
</ul>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-57915935404374189842011-12-24T17:11:00.003-08:002011-12-24T17:14:24.564-08:00TMB solution for 1ml - ELISA<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<span class="szoveg2">TMB solution for 1ml </span><span class="szoveg3"><cite>[10mg/ml TNB in DMSO]</cite></span><br />
<ul class="szoveg3">
<li>Disolve in 1ml DMSO {Dimethyl sulfoxide, minimum 99.5% GC Sigma D5879-100ml }</li>
<ul>
<li>10mg TNP {3,3',5,5'-tetramethyl-benzidine Sigma T-2885 FW240.3}</li>
</ul>
<li>Store at +4C</li>
</ul>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-54323135086487298482011-12-24T17:11:00.001-08:002011-12-24T17:14:24.561-08:00ELISA blocking-buffer for 10ml<div dir="ltr" style="text-align: left;" trbidi="on">
<br />
<span class="szoveg2">ELISA blocking-buffer for 10ml </span><span class="szoveg3"><cite>[0.05% Tween 20, 5%BSA, 0.05% azide, PBS]</cite></span><br />
<ul class="szoveg3">
<li>Mix on magnetic stirrer:</li>
<ul>
<li><span class="szoveg3">10ml PBS-Tween</span></li>
<li><span class="szoveg3">100mg BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V} </span></li>
<li><span class="szoveg3">50ul Sodium azide (from 10% NaN<sub>3</sub> stock solution)</span></li>
</ul>
<li>Store at +4C</li>
</ul>
</div>Unknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-44513184408708199052011-12-24T17:10:00.003-08:002011-12-24T17:14:24.555-08:00Carbonate buffer pH 9.5 for 1 liter (for ELISA coat)Carbonate buffer pH 9.5 for 1 liter (for ELISA coat)
Disolve in 1 liter Milli Q water:
1.6g Na2CO3
2.9g NaHCO3
Adjust to pH 9.5
Store at R/TUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-92029909101951011142011-12-24T17:10:00.001-08:002011-12-24T17:10:16.485-08:00ELISA MethodMethod
Coat ELISA plate with 50ul/well 0.2-10ug/ml protein (purity should be above 3%) in PBS or carbonate buffer for 2h on R/T or O/N in the fridge
Wash 3x with PBS-Tween (alternative: following washing incubate in ELISA-blocking buffer for 30min R/T and wash 3x with PBS-Tween)
Incubate in PBS-Tween diluted antibody/protein solution for 1h at 37C (ideally after 5h reach the equibrium)
Wash 3x with PBS-Tween
Develope with TMB
Add 100ul TMB developing solution to wells
For 1 ELISA plate mix:
11ml TMB buffer
110ul TMB solution
22ul H2O2 {Hydrogen peroxide 30% solution Sigma H-1009}
Add 100ul 2M H2SO4 to stop the reaction
Detect absorbance at 450nmUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-84376695277967537352011-12-24T17:09:00.005-08:002011-12-24T17:14:24.593-08:005x Veronal buffer for 500ml (5xVBS) Recipe5x Veronal buffer for 500ml (5xVBS) [727mM NaCl, 9.12mM Na-diethyl-barbiturate (C8H11O3N2Na), 15.63mM 5,5’-diethyl barbiture acid (C8H12N2O3), pH7.3]
Make solution 'A':
0.94g Na-diethyl-barbiturate (C8H11O3N2Na){5,5-Diathylbarbitursaure. Na-salz Serva 18797; MW:206.2}
21.25 g NaCl {Reanal, No.:2464-1-22-38, Nátrium-klorid, Ph. Eur.5; MW:58.44}
~300ml Milli Q water
Make solution 'B':
1.44g 5,5’- diethyl barbiture acid {5,5-dietil-barbitursav Reanal 04049; MW:184.2}
~100ml Milli Q water
Boil it otherwise it won’t dissolve, then cool it down
Mix solution ‘A’ and ‘B’ set pH to 7.3 with app. 50-70μl 10N NaOH
Fill up until 500ml with Milli Q water, then store it at +4CUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-41763078508009136592011-12-24T17:09:00.003-08:002011-12-24T17:14:24.551-08:001x Veronal buffer recipe1x Veronal buffer (VBS)[145mM NaCl, 1,8mM Na-diethyl-barbiturate (C8H11O3N2Na), 3mM 5,5’-diethyl barbiture acid (C8H12N2O3), pH7.3]
Dilute 5x Veronal buffer with Milli Q waterUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-67306217284697126962011-12-24T17:09:00.001-08:002011-12-24T17:14:24.571-08:000.2M EGTA stock solution for 10ml0.2M EGTA stock solution for 10ml
Mix on magnetic stirrer:
8ml Milli Q water
0.7608g EGTA{ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) Sigma E-4378 LOT 111K5411 FW 380,4}
Adjust to pH8 with 10N NaOH otherwise it won't disolve (be careful EGTA is an acid)
Adjust volume to 10ml with Milli Q water
Store at 4CUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-13255276217912475302011-12-24T17:08:00.007-08:002011-12-24T17:14:24.541-08:00EDTA-VBS solution for 10mlEDTA-VBS solution for 10ml (dilute serum 5x with it)[25mM EDTA, 5%BSA, 0.05% Tween 20, VBS ]
Mix on magnetic stirrer:
2ml 5x VBS
500ul 0.5M EDTA
0.5g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
50ul 10% Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
Adjust volume to 10ml with Milli Q water
Filtrate though an 0.45um filter
Aliquot 1ml/eppendorf
Store at -20CUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-74930827750137399532011-12-24T17:08:00.005-08:002011-12-24T17:14:24.584-08:00Mg-EGTA-VBS solution for 10mlMg-EGTA-VBS solution for 10ml (dilute serum 5x with it)[2.5mM Mg2+, 6.2mM EGTA, 5%BSA, 0.05% Tween 20, VBS]
Mix on magnetic stirrer:
2ml 5x VBS
310ul 0.2M EGTA
25ul 1M MgCl2 stock solution
0.5g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
50ul 10% Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
Adjust volume to 10ml with Milli Q water
Filtrate though an 0.45um filter
Aliquot 1ml/eppendorf
Store at -20CUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-12000213816309903592011-12-24T17:08:00.003-08:002011-12-24T17:14:24.537-08:00Ca-Mg-VBS (veronal buffered saline) for 25mlCa-Mg-VBS (veronal buffered saline) for 25ml (for serum dilution)[2.5mM Ca2+, 0.7mM Mg2+, 0.05% Tween 20, 5%BSA, VBS]
Mix on magnetic stirrer:
5ml 5x VBS
250ul Ca2+ Mg2+ stock solution [0.25M Ca2+, 0.07M Mg2+]
1.25g BSA {Sigma, No.:A3059-100G, Albumin, bovine serum, Fraction V}
12.5ul Tween 20 {Sigma No.:P-1379, Polyoxylethylene-sorbitan monolaurate (Tween 20)}
Adjust volume to 25ml with Milli Q water
Filtrate though an 0.45um filter
Aliquot 1ml/eppendorf
Store at -20CUnknownnoreply@blogger.com0tag:blogger.com,1999:blog-984222275347260670.post-62285360162943823472011-12-24T17:08:00.001-08:002011-12-24T17:14:24.568-08:00Ca2+ Mg2+ stock solution for 5ml [0.25M Ca2+, 0.07M Mg2+]Ca2+ Mg2+ stock solution for 5ml [0.25M Ca2+, 0.07M Mg2+]
Dissolve in 5 ml Milli Q water:
0.1838g CaCl2*2H2O {Reanal 11024 MSZ 24161-65 Kalcium-klorid, szárított MW: 147.02 CaCl2*2H2O}
0.0712g MgCl2*6H2O {Reanal No.: 20281-1-01-38 Magnézium-klorid 6-hidrát MW: 203.30 MgCl2*6H2O}Unknownnoreply@blogger.com0