Vector for TA cloning

June 11, 2015

BlueskyReddit
Osmania University
Department of Biochemistry
Research Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Promega

Product Name:

pGEM-T Easy Vector System

Catalog Number:

A1360

We use this kit for cloning genes. By using this kit, we can select specific pairs of restriction enzyme sites at both the 5' and 3' ends of the desired gene to clone into a vector of interest. Blue white screening can be done to select the colonies of a specific insert.

Experimental Design and Results Summary

Application

Cloning

Starting Material

Genomic DNA

Protocol Overview

We followed the manufacturer protocol. In short, 5 microliters of the 2x reaction buffer, 1 microliter of PGEMT vector (50ng/microliter conc), 1 microliter of the t4 ligase enzyme, and 3 microliters of insert were incubated at 4 degrees overnight. 2 microliters of the sample is transformed into the DHF-alpha strain bateria.

Tips

Use high quality insert DNA (Qiagen reaction clean-up kit )

Results Summary

We got a good number of white colonies with specific inserts.

Additional Notes

None

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Summary

The Good

Easy to identify insert-positive colonies by blue white screening and the vector has ampicillin resistance gene.

The Bad

One hour incubation did not give a higher number of insert-positive colonies

The Bottom Line

I recommend this vector for cloning.

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