Cloning with pGEM®-T Vectors: Ligation

One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems. This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase. T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert. The insert is directly ligated to the T-tailed plasmid vector with T4 DNA ligase. The insert can then be easily transferred from the T vector to other plasmids using the restriction sites present in the multiple cloning region of the T vector.

Proofreading polymerases like Pfu do not add “A” overhangs so PCR products generated with these polymerases are blunt-ended. In a previous blog, we discussed a simple method for adding an A-tail to any blunt-ended DNA fragment to enable T-vector cloning. Below, we think about the next step: Ligation.

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T-Vector Cloning: Questions, Answers and Tips

Blue/White colony screening helps you pick only the colonies that have your insert.

Q: Can PCR products generated with GoTaq DNA Polymerase be used to for T- vector cloning?

A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq® DNA Polymerase lacks 3’ →5’ exonuclease activity and displays terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerases (including the GoTaq® Flexi and GoTaq® G2 polymerases) will contain A-overhangs which makes them suitable for T-vector cloning with the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.

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The 5 Stages of Failed Cloning Grief (and how to get back on track!)

Cloning is a fickle process that can make even the most seasoned bench scientists scream in frustration. By the time you perform a colony PCR and run the gel to check for your insert, you’ve invested several days in preparing these transformed cells. But then, the unthinkable happens. When you image your gel…the target band is missing.

This can trigger what’s known as “The 5 Stages of Failed Cloning Grief.” As you work through each stage at your own pace, just know that scientists all over the world feel your pain and can empathize with you in this difficult time. Continue reading “The 5 Stages of Failed Cloning Grief (and how to get back on track!)”