I have isolated the mRNA and already made the cDNA now I face the problem of making the cDNA end sticky. Can anyone suggest me how to make the ends sticky for inserting into pmal plasmid?
You may need to design longer primers containing target sequence for a restriction endonuclease at their 5´ end. After the PCR of a cDNA template you digest your PCR product with these restriction enzymes. This will create sticky ends. Purified product can then be ligated into your vector. Please remember that the target sequence of a restriction enzyme should be at least 5 bp from the primer 5´ end. For example, the structure of a primer that is cut with BamHI is as follows: 5´-NNNNNGGATCCplus20nt of target-specific sequence-3´. After the digestion, the PCR fragment with have a 5´GATC/G-3´... overhang and can be cloned into the BamHI or BgLII site of the vector.
First of all, since cDNA is single stranded, you cannot make it sticky endy.. ;)
Second, Ales is correctly describing one way to go. It depends on what you want to do later with your amplicons.
Third, as long as you refer to a cloning strategy based on a PCR product and want to go on with your amplicons directly without having followed a particular strategy yet, you could also use a TA cloning vector. This system allows to clone any PCR product in the provided vector of the TA cloning system , which may serve (your choice, there are several TA vectors available) for cloning strategies (clike colony separation after transformation in bacteria) or protein expression or mRNA snthesis.
The principle behind is that Taq polymerase goes step by step while amplifying, and starts first hanging an adenosine nucleotide to the 3' end, which will be lateron substituted by the correct nucleotide corresponding to the second strand of DNA. If your amplicon ends, there is no corresponding strand and the 3'-terminal A stays for several days, because polymerase cannot find a corresponding base.
In other words, all PCR amplicons (as long as you do not use specific Taq polymerases (e.g.Vent polymerase)) creates a 3' A overhang, so to say a sticky end, which can be used for this specific cloning process.
Of note, the A is not necessarily original sequence of your target gene, but if necessary you can make it fitting by your primer choice (next base in 5' direction should be an T then).
Ales and Hauke thank you so much for the answers….can you guys kindly suggest which cloning kit to use in my case. I would like to express certain clones later….. i would be thankful..CHEERS!
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