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

PCR Cloning: Answers to Some Frequently Asked Questions

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.

Troubleshooting T-Vector Cloning

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.

A Quick Method for A Tailing PCR Products

Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome. Continue reading

Cloning Modified Blunt-ended DNA Fragments into T-Vectors

Tailing blunt-ended DNA fragments with TaqDNA Polymerase allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors. This method also eliminates some of the requirements of conventional blunt-end cloning — Fewer steps, who can argue with that?

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.

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