Cloning

Choosing Your Subcloning Strategy

Posted on by Kelly Grooms

Before you begin your subcloning, you need to know: The restriction enzyme (RE) sites available for subcloning in your parent vector multiple cloning region (or in the insert if you need to digest the insert); the RE sites available in the destination vector multiple cloning region (MCR); and if these same sites also occur in your insert. Once you know this information, you can use the chart below to decide which subcloning strategy to use.

4498MA-[Converted]

To learn more about subcloning, visit our Subcloning Notebook.

Get More Out of Your Lentiviral Production

Posted on by Promega

fugene6_lvv_blogThis review is a guest blog by Amy Landreman, Product Specialist in Cellular Analysis at Promega Corporation.

Lentiviral vectors (LVV) have become a valuable research tool for delivering genetic content into a wide range of cell types. Commonly derived from the HIV-1 genome, LVV have the advantage of being able to infect both dividing and non-dividing cells. They can be particularly valuable for introducing genetic material into cell lines that are difficult to transfect using other methods and are also being used in gene therapy applications.

Unlike other gene delivery tools, transducing mammalian cells with LVV requires significant upfront effort since the LVV particles carrying the desired genetic content first need to be created. In general this involves co-transfecting a packaging cell line, such as HEK293T, with a set of three to four separate plasmids that encode the protein content required to generate the LVV particles: the transfer plasmid, which contains the transgene of interest, a packaging plasmid, and an envelope plasmid. After co-transfection, the packaging cell line is allowed to incubate for a couple of days during which time the LVV particles are produced and accumulating in the culture supernatant. The supernatant containing the recombinant LVV is then harvested and, following several concentration steps, the LVV particles are ready to be used for introducing the desired genetic content into the mammalian target cells. Continue reading “Get More Out of Your Lentiviral Production”

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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