pcr cloning

Avoid the Cloning Blues This Season

Posted on by Michele Arduengo

I was blasting a holiday music playlist while driving recently, and Presley’s Blue Christmas played. I couldn’t get the phrase “Christmas Cloning Blues” out of my mind, and by the time I arrived at my destination, this happened:

Cloning Blues Christmas

(to the tune of Blue Christmas by Elvis Presley)

Blue and white colonies on a selective plate. Careful planning can help you avoid the cloning blues
Blue/White cloning is a standard technique in molecular biology labs.

I’ll have a blue Christmas without you

Colonies so blue, insert without you

Incubating my plates at 37 degrees

Won’t be the same if you’re not in lacZ

And all those blue colonies are forming

When my lab mates’ clonings are performing

They’ll be doing alright,

With their plates all filled with white

But I’ll have a blue, blue, blue cloning

Continue reading “Avoid the Cloning Blues This Season”

Cloning with pGEM®-T Vectors: Ligation

Posted on by Leah Cronan
T-Vector Cloning

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.

Continue reading “Cloning with pGEM®-T Vectors: Ligation”

Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.

Posted on by Leah Cronan

PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end. However, another thermostable DNA polymerase, like Taq DNA Polymerase, adds a single nucleotide base to the 3’ end of the DNA fragment, usually an adenine, creating an “A” overhang. This “A” overhang can create difficulties when cloning the fragment is your end goal. You might consider creating a blunt end with Klenow or adding restriction sites to the ends of your PCR fragment by designing them in your primers. But why go through all those extra steps, when that “A” overhang allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors? Fewer steps? Who can argue with that?

Continue reading “Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.”

Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors

Posted on by Leah Cronan

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

Continue reading “Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors”

T-Vector Cloning: Questions, Answers and Tips

Posted on by Leah Cronan
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.

Continue reading “T-Vector Cloning: Questions, Answers and Tips”

Characterizing Unique Protein: DNA Interactions Using Cell-Free Protein Expression

Posted on by Gary Kobs
Molecular model of human telomere DNA
Molecular model of human telomere DNA

The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5′-TTAGGGTTAG-3′ and forms a dimer with the TPP1 protein. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5′-TTAGGG whereas OB2 binds to the downstream TTAG-3′ (1,2). Several recent studies from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected (3). A recent reference reexamined the sequence-specificity of the Human POT1 protein (4).
SELEX (Systematic Evolution of Ligands through Exponential Enrichment) was used  to re-examine the DNA-binding specificity of human POT1 (5).

Continue reading “Characterizing Unique Protein: DNA Interactions Using Cell-Free Protein Expression”

T-Vector Cloning: Answers to Frequently Asked Questions

Posted on by Radhika Ganeshan
Blue/White colony screening helps you pick only the colonies that have your insert.
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 (proof reading) and also displays non-template–dependent terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning.

We have successfully cloned PCR products generated using GoTaq® and GoTaq® Flexi DNA Polymerases into the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.

Q: Can GoTaq® Long PCR Master Mix be used for T-Vector Cloning?

A: Yes it can. GoTaq® Long PCR Master Mix utilizes recombinant Taq DNA polymerase as well as a small amount of a recombinant proofreading DNA polymerase. This 3´→5´ exonuclease activity (proof reading) enables amplification of long targets. Despite the presence of a small amount of 3´→5´ exonuclease activity, the GoTaq® Long PCR Master Mix generates PCR products that can be successfully ligated into the pGEM®-T Easy Vector System.

We have demonstrated that GoTaq® Long PCR Master Mix successfully generated DNA fragments that could be ligated into pGEM®-T Easy Vector System without an A-tailing procedure, and with ligation efficiencies similar to those observed with the GoTaq® Green Master Mix.

For details refer to Truman, A., Hook, B. and Wieczorek, D. Using GoTaq® Long PCR Master Mix for T-Vector Cloning.

Tip: For cloning blunt-ended PCR fragments into T-vectors, use the A-tailing protocol discussed in the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

Q: How do I prepare PCR products for ligation? What products can be used to purify the DNA?

Continue reading “T-Vector Cloning: Answers to Frequently Asked Questions”

PCR Cloning: Answers to Some Frequently Asked Questions

Posted on by Kelly Grooms

eh1Q: What is the easiest way to clone PCR Products?

A: The simplest way to clone PCR Products is to amplify the product using thermostable polymerases such as Taq, Tfl or Tth polymerase. These polymerases add a single deoxyadenosine to the 3´-end of the amplified products (3´-end overhang), and can be cloned directly into a linearized T-vector.

Q: What if my DNA polymerase has 3´ to 5´ exonuclease activity (i.e., proofreading activity) that removes the 3´-end overhang?

A: To clone PCR products that have been amplified with a polymerase that have proof reading activity into a T-vector, you will need to perform an A-tailing step using Taq DNA polymerase and dATP. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.

Set up the following reaction in a thin-walled PCR tube:

1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
1µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl

Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.

Q: What is a T-vector, and why are they used for cloning PCR products?

A: T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.

For a complete PCR Cloning protocol, Visit the Cloning Chapter of the Promega Protocols and Applications Guide.

Successful Ligation and Cloning of Your Insert

Posted on by Sara Klink
Ligation and cloning
Cloning PCR product.

You have PCR amplified your insert of interest, made sure the PCR product is A tailed and are ready to clone into a T vector (e.g., pGEM®-T Easy Vector). The next step is as simple as mixing a few microliters of your purified product with the cloning vector in the presence of DNA ligase, buffer and ATP, right? In fact, you may need to consider the molar ratio of T vector to insert.

Continue reading “Successful Ligation and Cloning of Your Insert”

Troubleshooting T-Vector Cloning

Posted on by Promega

Why do few of my pGEM®-T or pGEM®-T Easy Vector clones contain the PCR product of interest?

There are several possible reasons why the PCR product may not be recovered after ligation, bacterial transformation and plating when using the pGEM®-T or pGEM®-T Easy Vector Systems.

The PCR fragment may not be A-tailed. Without the A overhangs, the PCR product cannot be ligated into a T vector. Use a nonproofreading DNA polymerase like GoTaq® DNA Polymerase for PCR. If a proofreading DNA polymerase is used, A overhangs will need to be added. Purify the PCR fragment, and set up an A-tailing reaction (see the pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual #TM042). The A-tailed product can be added directly to the ligation as described in the pGEM®-T or pGEM®-T Easy Vector protocol.

The insert:vector ratio may not be optimal. The ideal ratio for each insert to a vector can vary. For example, the Control Insert DNA works well at a 1:1 ratio, but another insert may be ligated more efficiently at a 3:1 ratio. Check the integrity and quantity of your PCR fragment by gel analysis. Optimize the insert:vector ratio (see Technical Manual #TM042).

Multiple PCR products were amplified and cloned into the pGEM®-T or pGEM®-T Easy Vector. Other amplification products including primer dimers will compete for ligation into the T vector, decreasing the possibility that the desired insert will be cloned. To minimize other competing products, gel purify the PCR fragment of interest.

Promega Technical Services Scientists are here to assist you in troubleshooting your experiments at any time. Contact Technical Services.

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