What is the difference between the pCR2.1-TOPO and pCR4-TOPO vectors?
The vector backbones for both of these vectors are very similar. The main difference is that the pCR4-TOPO vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR4-TOPO vector very useful for sequencing applications.
What is the difference between the pCR2.1-TOPO and pCRII-TOPO vectors?
The vector backbones for both of these vectors are very similar. The main difference is that the pCRII-TOPO vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR2.1-TOPO vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.
Your TOPO cloning kits contain a control template and control primers. Can I obtain the sequence of the control template?
The sequence of the control template is proprietary.
How can I increase the intensity of the crystal violet-stain in my gel?
To increase the intensity of the crystal violet-stained gel, allow the gel to lie for a couple of hours in crystal violet stain (45 µL of 2 mg/mL stain in 100 mL sterile water or 1-10 µg/mL stain in 0.1X TAE) and it will stain a little darker. Place the gel over a white background when excising the band to improve visibility.
Which of the following products will usually give the best cloning efficiency in terms of positive clones and number of colonies: Zero Blunt PCR, Zero Blunt PCR-TOPO, or Zero Blunt PCR-TOPO for Sequencing?
Typically 200-300 colonies are obtained for the TOPO kits with 100% inserts (20/20). For the Zero Blunt kit that uses standard ligase, typically 600-1000 colonies are obtained with 95-100% inserts.