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Cloning pDsRed-Monomer-N1


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#1 Raekah

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Posted 19 March 2014 - 05:21 AM

Hi,

 

 I'm trying to clone a small insert into this vecotr. Double digested both the vector and insert with BamH1 and PST1, Gel purified and ligated with T4 DNA Ligase using JM109 cells. No colonies. Have tried it about 5 times. No idea wha to do :(



#2 jerryshelly1

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Posted 19 March 2014 - 11:15 AM

It would be better if you could provide a detailed description of what you are doing. There are tons of cloning resources on this website that you might consider browsing through. My most common cause of ligation failure is proper digestion of my PCR fragments. You usually get good digestion with a vector due to its size and shape, but PCR fragments can be problematic depending on the size.

 

I usually like to test the efficiency of my ligation by running a simple PCR with a primers that recognize both the vector and PCR fragment. Amplification is a good indication that some ligated products are in your sample.

 

What are your JM109 cell controls like? Do you observe a good transformation efficiency?






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