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How to avoid contamination in PCR?

Oct 02 2004 12:24 PM | bioforum in PCR

PCR contaminations are like ghosts... you know they are there but you dont know WHERE?? I have followed the following with good results... Prepare your master mix in a separate room from your current location, somewhere PCR is not the main practice....

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How to convert g into rpm?

May 28 2009 07:05 PM | bioforum in Centrifugation

The relationship between revolutions per minute (RPM) and relative centrifugal force (xg) is: g = (1.118 × 10-5) R S2 where g is the relative centrifugal force, R is the radius of the rotor in centimeters, and S is the speed of the centrifuge in revolu...

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How to get rid of PCR primer dimer

Jan 29 2005 08:06 PM | bioforum in RT-PCR

1) Try the DMSO up to 5%. Some polymerases are ok with DMSO and others aren't. You never know until you try. 2) Try a two step PCR. There is a paper in Biotechniques about a two step PCR reaction but I don't have the reference with me sin...

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Handling RNA - How to avoid RNase contamination?

Dec 08 2009 09:31 PM | bioforum in RNA Methods

Hi Susan, I would second what tea-test has pointed out. Any tissue you process is going to be loaded with RNases (cells contain RNases). Any residual RNase present on your homogenizer is going to be very minor compared to what is already present in...

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How to coat coverslips before seeding cells?

Aug 18 2011 05:55 AM | jennycharlies in Cell Culture

jennycharlies Posted 18 August 2011 - 06:55 AM Hi, I am going to seed my cells onto coverslips inside the wells of a 12 well plate and was wondering what is the best way to coat the coverslips? I have 18mm coverslips and need to coat with poly-D-...

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Can 18s gene be used as reference gene for RT-PCR?

Mar 10 2013 06:31 PM | memari in RT-PCR

QuoteHi, I have done few real time PCR works recently, and I used the 18s gene for endogeneous control. Its Ct value was approximately 11 for every cDNAs used as template. Here, I have a question. Since 18s gene doesn't have poly A tail, it can...

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How to find promoter sequence for methylation s...

Aug 05 2009 07:43 PM | pcrman in Bioinformatics and Biostatistics

Many people have problem identifying or predicting the promoter sequence of a gene, or don't know how to get the actual sequence for analysis such as primer design, transcription factor binding site search, etc. Here I provide ways how I do these t...

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Sonication Vs MNase for ChIP assay

Apr 14 2011 09:59 AM | Dukey in ChIP Assay

I am sure that this debate has been covered before, but I just wanted to share my own personal experiences with it. I am working with a TF and fairly recently switched to MNase. I initially tried MNase digestion out of pure curiousity, but I have to sa...

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DNA migrate differently in agarose vs PAGE gel

Aug 15 2011 08:59 PM | pcrman in Electrophoresis

Quotei am currently working a gene in leukemia. The mutant allele has been sequenced to contain 527 base pair. when i run an agarose gel to confirm, it did appear in the position approximately equal to 527 base pair according to the DNA ladder. HOWEVER...

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Tips on freezing and thawing of cells

Sep 10 2004 12:32 PM | bioforum in Cell Culture

I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree. Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500...

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