FAQs

GoTaq^® and GoTaq^® Flexi DNA Polymerase

See Technical Resources for more information.

1. What are the GoTaq^® and GoTaq^® Flexi DNA Polymerases?
2. Why are two reaction buffers supplied with GoTaq^® and GoTaq^® Flexi DNA Polymerases?
3. What type of gel can I use to separate PCR products amplified using GoTaq^® or GoTaq^® Flexi DNA Polymerase?
4. Do the dyes in the green GoTaq^® reaction buffers affect migration of DNA on agarose gels?
5. What DNA purification methods are compatible with GoTaq^® and GoTaq^® Flexi DNA Polymerases?
6. Can I use GoTaq^® or GoTaq^® Flexi DNA Polymerase in RT-PCR with Promega ImProm-II™ Reverse Transcriptase or the Reverse Transcription System?
7. Can I use GoTaq^® and GoTaq^® Flexi DNA Polymerases with hot-start antibodies such as the TaqStart™ antibody?
8. Can I clone PCR products generated using GoTaq^® or GoTaq^® Flexi DNA Polymerase successfully into T-vectors?

1. What are GoTaq^® and GoTaq^® Flexi DNA Polymerases?
   GoTaq^® DNA Polymerase and GoTaq^® Flexi DNA Polymerase are a formulation of native Taq DNA polymerase for all routine PCR applications. GoTaq^® DNA Polymerase is supplied with two reaction buffers, each of which contains MgCl₂ at a final concentration of 1.5mM in the amplification. GoTaq^® Flexi DNA Polymerase is supplied with two Flexi reaction buffers, which do not contain MgCl₂, and a separate vial of 25mM MgCl₂ so that Mg²⁺ concentration can be optimized or adjusted as needed.

2. Why are two reaction buffers supplied with GoTaq^® and GoTaq^® Flexi DNA Polymerases?
   GoTaq^® DNA Polymerase is supplied with the 5X Green GoTaq^® Reaction Buffer and 5X Colorless GoTaq^® Reaction Buffer. The GoTaq^® Flexi DNA Polymerase is supplied with Mg²⁺-free 5X Green GoTaq^® Flexi Buffer and 5X Colorless GoTaq^® Flexi Buffer. DNA amplified using the green reaction buffers can be loaded directly onto agarose gels, eliminating the need to add loading dye or buffer to the PCR sample before electrophoresis. When diluted to 1X, the green reaction buffers have sufficient density to sink DNA samples into the wells of an agarose gel or nondenaturing TBE polyacrylamide gel. Also, the green reaction buffers contain two dyes, a blue dye and a yellow dye, which separate upon electrophoresis and can be used to monitor progress of the DNA on the gel. The blue dye migrates at the same rate as a 3–5kb DNA fragment in a 1% agarose gel. The yellow dye migrates at a rate faster than that of the amplification primers (<50bp).
   
   The blue and yellow dyes in the green reaction buffers can interfere with applications requiring absorbance or fluorescence measurements. These dyes absorb light between 225nm and 300nm, making standard A₂₆₀ readings to determine DNA concentration unreliable. The dyes also have excitation peaks at 488nm and 600–700nm, which correspond to the excitation wavelengths of commonly used fluorescence-detection instrumentation. The colorless reaction buffers are provided for use with GoTaq^® or GoTaq^® Flexi DNA Polymerase when performing applications requiring absorbance or fluorescence measurements without prior purification of the PCR product. However, the green reaction buffers also can be used for PCR prior to such applications if the DNA fragments are purified first.

3. What type of gel can I use to separate PCR products amplified using GoTaq^® or GoTaq^® Flexi DNA Polymerase?
   In addition to standard agarose gels, we have been able to get high resolution of PCR products on nondenaturing TBE acrylamide gels as well as Invitrogen's E-gel^® (2% agarose). GoTaq^®-amplified samples can also be run on gels containing ethidium bromide, with no effect on migration. However, incorporation of SYBR^® GOLD into the agarose gel prior to electrophoresis can interfere with visualization of PCR products amplified using GoTaq^® or GoTaq^® Flexi DNA Polymerase and Green GoTaq^® Reaction Buffer or Green GoTaq^® Flexi Buffer, respectively. This may be due to nonspecific interaction of SYBR^® GOLD with the blue or yellow dyes; however, this can be overcome by staining the gel after electrophoresis is complete.

4. Do the dyes in the green GoTaq^® reaction buffers affect migration of DNA on agarose gels?
   The dyes do not interfere with migration of DNA in agarose gels; fragments migrate the same distance as corresponding DNA markers. There are no differences in the migration pattern of DNA samples containing green reaction buffers, samples without dye, or samples containing Promega Blue/Orange Loading Dye (Cat.# G1881).

5. What DNA purification methods are compatible with GoTaq^® and GoTaq^® Flexi DNA Polymerases?
   The Wizard^® SV Gel and PCR Clean-Up System (Cat.# A9281) and Wizard^® PCR Preps DNA Purification System (Cat.# A7170) can be used to purify PCR fragments generated using GoTaq^® or GoTaq^® Flexi DNA Polymerase. The QIAquick™ PCR clean-up system (Qiagen), ethanol precipitation or gel-purification can also be used to purify PCR fragments amplified with GoTaq^® or GoTaq^® Flexi DNA Polymerase. The dyes in the 5X Green GoTaq^® Reaction Buffer and 5X Green GoTaq^® Flexi Buffer are efficiently removed by all of these purification methods as determined by absorbance readings in the range of 200–700nm both before and after purification of amplified PCR products. The absorbance peaks associated with the two dyes are not detected in absorbance scans of PCR products after purification.

6. Can I use GoTaq^® or GoTaq^® Flexi DNA Polymerase in RT-PCR with Promega ImProm-II™ Reverse Transcriptase or the Reverse Transcription System?
   Yes. We have amplified cDNA generated in a 20μl ImProm-II™ (Cat.# A3801) reverse transcription reaction using PCR (100μl volume) and GoTaq^® or GoTaq^® Flexi DNA Polymerase. Comparable results were obtained using the green and colorless reaction buffers. For both enzymes, sensitivity down to 10 copies of beta-actin RNA was obtained in amplifications with total mouse liver RNA. GoTaq^® and GoTaq^® Flexi DNA Polymerases are also compatible with the AMV RT-based Reverse Transcription System (Cat.# A3500).

7. Can I use GoTaq^® or GoTaq^® Flexi DNA Polymerases with hot-start antibodies such as the TaqStart™ antibody?
   We have used GoTaq^® and GoTaq^® Flexi DNA Polymerases in both the colorless and green reaction buffers successfully in hot-start PCR using the TaqStart™ antibody (Clontech).

8. Can I clone PCR products generated using GoTaq^® or GoTaq^® Flexi DNA Polymerase successfully into T-vectors?
   Yes. We have successfully cloned PCR products generated using GoTaq^® and GoTaq^® Flexi DNA Polymerases into the pGEM^®-T (Cat.# A3600), pGEM^®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors after purification of the amplimer to remove unincorporated primers. The Wizard^® SV Gel and PCR Clean-Up System (Cat.# A9281) can be used to purify GoTaq^® Polymerase-generated PCR products prior to T-vector cloning. Also we have successfully cloned PCR products directly without prior purification in cases where a single amplification product is generated. However, even if no extraneous bands are visible, there may be some primer-dimers present in the reaction, and this can result in unacceptably high numbers of clones containing primer-dimer instead of the fragment of interest.