How do you prep your cDNAs?

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ehd12
ehd12's picture
How do you prep your cDNAs?

Hi...

I'm patching Cav2.2 channel expressed in tsA201 cells.

I have problems in current size and success rate. 
Usually I get only one suitable current size (>0.5nA) in 10 whole cells. Also most of the cells have current near 100pA.
I already checked concentration, 260/280 and 260/230 ratio and they are in adequate range. 
However, one of my colleague who also studies calcium channels, examined my cDNAs (alpha1 and others) and he concluded that my cDNA is somewhat wrong!! 

I use miniprep for preparing cDNAs, but someone says  that cDNAs for transfection might be prepared by midi or maxi prep due to higher endotoxin level. So I guess that it can be the source of problem. 

So, do you use miniprep for prep cDNAs? And.. can higher endotoxin level cause small current?

Jason King
Jason King's picture
 Remember that cDNA stands

 Remember that cDNA stands for "copy" DNA and is made from mRNA using a reverse transcriptase. 

Plasmid DNA is made using bacterial preps and if you plan to transfer these plasmids they need to be as pure as possible. OD 260/280 between 1.80 and 1.90

being free of endotoxins also helps so you would use an endo-free kit (mini, midi, maxi, it doesn't matter as this refers to the quantity that can be purified). The gold standard is still Qiagen endo-free preps, unless you want to go back to purifying over two rounds of CsCl density centrifugation (no one wants to do this).

MUTHUMARIAPPAN ...
MUTHUMARIAPPAN Murugan's picture
Hi,

Hi,

Pls refer the link.. Protocol is available.

http://www.lsa.umich.edu/ummz/mollusks/labs/tfduda/pdf/cdna-prep.pdf

Regards
Muthu