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cDNA library construction


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#1 Bin

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Posted 08 April 2010 - 01:56 PM

Now I'm trying to make a cDNA library using clontech creator smart cDNA library construction kit. I bought a muscle mRNA from clontech,too.
I followed the protocol that clontech provides, but finally I get only one colony. It's not a library. I repeated it three times. ever try to increase the mRNA template or do ligation directly without Size Fractionation. The results are same, just several colonies.
Could someone tell me what's wrong with this?
I'm so frustrated about this.
Bin

#2 phage434

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Posted 08 April 2010 - 03:18 PM

Are you using competent cells supplied with the kit? Have you checked the competencies of these cells? Dilute a test circular plasmid to a concentration of 10 pg/ul. Transform your cells with 1 ul of this solution. You should have hundreds of colonies on the plate.

#3 Bin

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Posted 19 May 2010 - 11:14 AM

Are you using competent cells supplied with the kit? Have you checked the competencies of these cells? Dilute a test circular plasmid to a concentration of 10 pg/ul. Transform your cells with 1 ul of this solution. You should have hundreds of colonies on the plate.

Thank you! I'll try to check the competent cells.




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