Successful Ligation and Cloning of Your Insert

Ligation and cloningYou 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.

After the insert DNA is prepared for ligation, estimate the concentration by comparing the staining intensity of your PCR product with that of DNA molecular weight standard of similar size and known concentrations on an ethidium bromide-stained agarose gel. If the vector DNA concentration is unknown, estimate the vector concentration by the same method. Test various vector:insert DNA ratios to determine the optimal ratio for a particular vector and insert. In most cases, a 1:1 or 1:3 molar ratio of vector:insert works well, but you may want to consider 1:5, 5:1 and even a 10:1 ratio. The following example illustrates the calculation of the amount of insert required at a specific molar ratio of vector:insert.

[(ng of vector × kb size of insert) ÷ kb size of vector] × (molar amount of insert ÷ molar amount of vector) = ng of insert

Example:
How much 500bp insert DNA needs to be added to 100ng of 3.0kb vector in a ligation reaction for a desired vector:insert ratio of 1:3?
[(100ng vector × 0.5kb insert) ÷ 3.0kb vector] × (3 ÷ 1) = 50ng insert

Our BioMath Calculator is an easy way to calculate the molar ratio of vector to insert for ligation.

The vector:insert ratio changes, depending on the insert, even if you use the same vector. If you use the same vector:insert ratio for many different inserts and the insert size increases or decreases, recalculate the amount of insert needed for ligation using the equation above or our handy BioMath Calculator to ensure the molar ratio stays the same.

For more information on cloning, consult the Cloning chapter of the 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.

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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