gulash

As Roche doeasn't specify the sequences of it's LNA probes from universal probe library, and they are available when designing an assay I propose to make a list of them online (they often work even when the online tool say they don't:). May be helpful

I've got the sequence of
#3   CTGCTGGG    
#68  CTGCTCCT

88 to go:)

I've cloned a 1700 PCR product into a pJet vector - went perfectly allright. I want to transfer the insert into a pCMV5. The insert itself have EcoRI at the beging and BglII restriction site at the end. I double digest the pJet-Insert plasmid and ive got perfect bands on the gel. I isolate the band corresponding to the insert and clean it up with Axygen gel extraction kit. I double digest the pCMV5 vector and dephosphorylate it with TSAP, then clean it up with axygen pcr cleanup kit. I take 50 ng of digested and dephosphorylated vector and 3,4,5 times more (molar) of the digested insert, then ligate it with NEB quick ligase, directly transform the NEB DH5a competent cells ( full protocol) and spread them on LB-amp agar. Overnight i always get around 20 colonies per plate. After an hour outgrowth in LB i make a colony pcr for the insert and always analyse ALL the colonies (around 60 from all the plates)! Ive tried it 4 times now, and ive got NO insert whatsoever!! WHAT AM I DOING WRONG couse i'll go insane in the moment!

I've doube digested my pCMV5 vector with EcoRI an BglII, and as I've got some problem with it self-ligating, i've dephosphorylated it with CIAP. Now, do I need my ligase buffer to be free of ATP?? ( the insert is still phosphorylated after the same double digestion)