memari

It is a DNA contamination.
18s is not a good reference gene, because:

1) It has multiple copies in genome. 300 to 400 copies, thus they are much more abundant than target mRNA transcripts, which are usually rare. Thus:

2) You have to dilute sample for 18s as 1:6000 while you dilute sample for other gene 1:20 to bing its CT value between 20-30. Thus:

3) 18s is diluted much more than that of other genes, so it has less inhibitors because of more dilution.

4) It has pseudogenes.
https://www.roche-ap...notes/lc_15.pdf

Check it yourself: goto Primer-Blast website and past both forward and reverse primers in that website and click "Get Primers" buttom.
https://www.ncbi.nlm...s/primer-blast/

You will see that it is not possible to design a pair of primers for 18s that do not bind to more than one place.

5) Ribosomal RNA is resistant to degradation much more than mRNA.


Thus use Ribosomal Proteins instead of 18s.   (edited)
http://openwetware.o...R_normalisation

and my suggestion based on my experience:
If you can not find a gene (except 18s )with stable CT value in order to use it as a reference gene, it is your fault.
Because 100% I am sure that you have not done RT, RNA normalization and sampling very well.

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Babak

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Which antibody is good for HA-tag? I used santa cruz and cell signaling but both of them give alot of background bands.

What is the purpose of adding NaAc in Nucleic Acid Extraction?
http://www.protocol-...posts/3846.html

http://www.genetics....ls_Miniprep.htm

This Plasmid Purification is based on the small size and supercoiled nature of plasmid DNA.
EDTA causes instability in Membrane.
SDS solubilises the proteins and lipids of membrane which ends to disrupture in membrane of bacteria.
When you change the pH of solution with adding NaOH to 12-12.5 pH, Hydrogen Bands in Chromosomal DNA break and two strands of DNA are seperated.
But DNA of Plasmid stays intact because of the supercoiled nature of it.
After adding Acetat, Plasmid stays soluble but Chromosomal DNA precipites.
Plasmid precipites after adding IsoPropanol.
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Babak Memari

We have used this and now we have 5 fold more DNA for QPCR for Chip. It is definitly better tha QIAGEN.
Feldan's PCR Purification Kit
http://www.feldan.co...on-kit-200preps


Babak

Hi
I have collected all softwares that others have written here in my wordpress site:
https://babakmemari....lysis-software/

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Babak