Focus: T-Vector Cloning

Shorten the Ligation Time for the pGEM®-T Vector Systems

The pGEM®-T Vector Systems are convenient for cloning PCR products. To increase convenience, we tested conditions for shortening the ligation time. A 15-minute ligation gave ~50% transformants by blue/white selection with further improvements when BSA was added.

By Adam Petterson and Trista Schagat, Ph.D.
Promega Corporation

Published in January 2009

Introduction

With advances in cloning technologies, a researcher can easily and quickly create plasmids containing open reading frames (ORFs), genes or DNA segments for downstream assays, such as protein expression and sequencing. Many modern PCR techniques rely upon thermostable Taq DNA polymerase. Taq polymerase lacks 3´ to 5´ exonuclease (proofreading) activity; this results in amplified sequences with a deoxyadenosine overhang on the 3´ end. To exploit this feature, linearized plasmids called T vectors have been created with terminal 3´ thymidine residues on either end. These vectors are optimized for base pairing with the deoxyadenosine overhangs on PCR products from nonproofreading polymerases.

Promega offers several T-vector cloning solutions including the pGEM®-T (Cat.# A3600) and pGEM®-T Easy (Cat.# A1360) Vector Systems. Unlike cloning systems that use topoisomerases for expedited ligation, the pGEM®-T and pGEM®-T Easy Vectors require no enzyme modification. Instead, a proprietary 2X Rapid Ligation Buffer is included with both systems. The use of the 2X Rapid Ligation Buffer reduces the ligation incubation time from overnight to 1 hour. Here we test the ability to shorten the ligation time even further.

Shorten Ligation Time

The recommended ligation time in the pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual #TM042 is 1 hour at room temperature. Using the Control Insert DNA, we tested shorter ligation incubation times (Table 1). Using a 15-minute incubation time, 48% of transformants were obtained as determined by blue/white screening. The total number of colonies was reduced with shortened ligation times, but the numbers were sufficient for insert screening. The ability to obtain transformants after a shortened ligation time is consistent with a previous study (1).

Table 1. The Effect of Incubation Time on Ligating the Control Insert DNA into the pGEM®-T Vector. Ligation reactions were carried out following the protocol in TM042 using the 542bp Control Insert DNA provided with the vector system and the incubation times listed below, transformed into high-efficiency competent JM109 cells, then plated on agar containing 100µg/ml ampicillin, 0.5mM IPTG, and 80μg/ml X-Gal for blue/white screening. Plates were then incubated at 37°C overnight, and the number of blue and white colonies was scored. Data are the average of 2–6 replicates.
Ligation Time Total Colonies White Colonies (%)
1 hour 201 82
30 minutes 195 66
15 minutes 78 48

We then investigated using the 15-minute incubation time for ligation with longer sequences. A 1.1kb fragment of IL1-β was amplified from Mouse Genomic DNA (Cat.# G3091), and a 3.0kb fragment of the APC gene was amplified from Human Genomic DNA (Cat.# G3041) using GoTaq® DNA Polymerase. PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281) then ligated into the pGEM®-T Vector. After a 15-minute ligation incubation, these larger inserts gave >100 white colonies for screening (Table 2).

Table 2. The Effect of Insert Size on pGEM®-T Vector Ligation When Incubated for 15 Minutes. Two inserts (1.1kb and 3.0kb) were amplified by GoTaq® DNA Polymerase, gel purified and ligated into the pGEM®-T Vector by incubating for 15 minutes at room temperature. Data are the average of 2 replicates.
Insert Size Total Colonies White Colonies (%)
1.1kb 216 66
3.0kb 509 37

Addition of Bovine Serum Albumin (BSA)

Acetylated BSA is reported to confer stability to T4 DNA ligase (2). The activity of the enzyme increases substantially with temperature, resulting in decreased stability and hindered catalytic efficiency. BSA helps to sequester and stabilize the enzyme, as well as inhibit nonspecitic binding to reaction tubes, allowing for a substantial increase in enzyme stability and resulting in increased catalytic efficiency with a constant amount of substrate.

We tested the ability of BSA to improve results for the 15-minute ligation reaction. One microliter of 10mg/ml acetylated BSA (Cat.# R3961) was substituted for 1µl of water in each ligation. This is the same high-quality BSA preparation included with Promega restriction enzymes. The addition of BSA to the 15-minute ligation reaction increased the number of white colonies for all insert sizes tested
(Table 3). BSA did not significantly enhance the ligation of the control insert (0.5kb) at 1 hour and only had a modest effect at 30 minutes (data not shown).

Table 3. The Effect of BSA on pGEM®-T Ligation When Incubating for 15 Minutes. Two inserts, 1.1kb and 3.0kb, were amplified by GoTaq® DNA Polymerase and gel purified. The 0.5kb Control Insert DNA and the purified amplimers were added to the pGEM®-T Vector, mixed with 1µl of 10mg/ml acetylated BSA (Cat.# R3961) and ligated for 15 minutes at room temperature. Data are the average of 2–4 replicates. The column "Change in White Colonies with BSA" shows the difference between the percentage of white colonies achieved when ligation was performed with and without BSA (Tables 1 and 2).
Insert Size Total Colonies % White Colonies Change in White Colonies with BSA
0.5kb 159 63 +15%
1.1kb 185 74 +8%
3.0kb 529 51 +13%

Summary

This study showed that the incubation time for pGEM®-T Vector ligations can be shortened to 15 minutes at room temperature. To increase the number of recombinants obtained, we recommend adding high-quality BSA to a final concentration of 1µg/µl in the reaction.

References

  1. Frackman, S. and Kephart, D. (1994) Rapid ligation for the pGEM®-T and pGEM®-T Easy Vector Systems. Promega Notes 71, 8–9.
  2. Yoshino, Y., Ishida, M. and Horii, A. (2007) A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the "Coffee Break Ligation" technique. Biotechnol. Lett. 29, 1557–60.