Amplification and Purification
We used the pGL4.10[luc2] Vector (Cat.# E6651) to amplify the luciferase reporter gene luc2 (1,652bp) using the forward primer: 5´-TGGTGCGATCGCCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAG-3´, the reverse primer: 5´-GTGAGTTTAAACCACGGCGATCTTGCCGCCCT-3´ and three different enzyme mixes:
- GoTaq® Long PCR Master Mix (Cat.# M4021)
- GoTaq® Green Master Mix (Cat.# M7122)
- PfuUltra Hotstart DNA Polymerase (Agilent Cat.# 600390)
The GoTaq® Green Master Mix is expected to generate A-tailed PCR fragments that can be cloned into the pGEM®-T Easy Vector. PfuUltra is expected to remove A-overhangs from the PCR product due to its proofreading ability.
We amplified luc2 using GoTaq® Long PCR Master Mix according to the protocol (TM359) with the suggested parameters (Table 1) on an MJ Research PTC-200 Thermal Cycler.
We also amplified the luc2 gene using the PfuUltra Hotstart DNA Polymerase and GoTaq® Green Master Mix as described in the PfuUltra Hotstart DNA Polymerase instruction manual. Both reactions were amplified on the Bio-Rad DNA Engine® Thermal Cycler (Table 2).
uFollowing amplification, we pooled three reactions from each enzyme mix for further analysis. The pooled PCR products were run on a 0.8% agarose gel and DNA was purified from gel slices using the Wizard® SV Gel and PCR Clean-up System (Cat.# A9281), and the concentration and purity of the cleaned-up PCR products were determined by NanoDrop®-1000 spectrophotometry. Finally, we visualized the purified products on a 0.8% agarose gel.
Ligation and Transformation
We ligated with the amplified luc2 DNA generated from PfuUltra Hot Start DNA Polymerase, GoTaq® Green Master Mix, and GoTaq® Long PCR Master Mix reactions into the pGEM®-T Easy Vector using a 3:1 insert to vector ratios according to the protocol (#TM042). The resulting pGEM®-T-luc2 clones were transformed into JM109 High Efficiency Competent Cells (>108cfu/µg; Cat.# L2001). Following transformation, the cells (100µl) were plated in triplicate onto LB plates with ampicillin/IPTG/X-Gal and incubated for 18 hours at 37°C. We inoculated liquid cultures with single white colonies and grew them overnight in LB with ampicillin. We used the PureYield™ Plasmid Miniprep System (Cat.# A1223) to isolate the plasmid DNA. To confirm the expected size of the DNA insert, we digested the purified plasmid DNA with EcoRI (Cat.# R6011) according to the Restriction Enzyme Protocol Technical Manual #TM367 and visualized the products on a 0.8% agarose gel.
Competent E. coli Comparison
To compare the efficiency between competent cell types, we transformed both the Pro 5-alpha Competent Cells (>109cfu/µg; Cat.# L1221), JM109 Competent Cells (>107cfu/µg) and JM109 High Efficiency Competent Cells (>108cfu/µg) with ligation reactions from the GoTaq® Long PCR reaction. Fifty microliters of each competent cell type was transformed according to the pGEM®-T Easy Vector System Technical Manual #TM042 and added to 950µl of SOC medium. We plated 100µl of each transformation mix (10% of total) in duplicate onto LB plates with ampicillin/IPTG/X-Gal, and used blue/white screening to determine colony counts.