Direct Cloning of DNA Inserts to Plasmid Vector

February 05, 2020

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

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Quality of Results

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Company:

Invitrogen

Product Name:

The Original TA Cloning Kit

Catalog Number:

45-0046

I used DNA inserts amplified using Taq DNA polymerase to directly clone into pCR2.1 linearised plasmid vector which comes with The Original TA cloning kit to construct vector called pAJ14. Used 100 ng of PCR products (directly) to ligate with vector in the TA cloning kit. Transformed the 5 microliter ligation mix into One Shot Top10 chemically competent E. coli cells. More than 70 percent of cells gave expected plasmid propagation.

Experimental Design and Results Summary

Application

Direct cloning DNA inserts amplified by Taq polymerase into a plasmid vector

Starting Material

100 ng PCR reaction amplified by Taq polymerase

Tips

None

Results Summary

Using The Original TA cloning kit, I used Taq DNA polymerase amplfied DNA inserts directly into pCR2.1 vector (linearised) in the kit. Followed easy -to-follow instructions in the kt. Finally transformed 5 microliter of ligation mix into One Shot Top10 chemically competent E. coli cells. Transformants were selected for kanamycin resistance marker and confirmed by PCR.

DOI or PMID #

N/A

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Summary

The Good

Fast easy cloning of DNA inserts, no enzymatic modification of inserts, no purification of PCR template required, and saves a lot of time.

The Bad

None

The Bottom Line

Ideal kit for fast cloning of DNA inserts into a plasmid vector and in several plasmid constructions.

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