What protein yields should I expect to get with the pRSET expression vectors?
What is the source of the EM7 promoter?
Where is the transcriptional start site of the T7 promoter featured in many of Invitrogen™'s vectors?
Although Invitrogen™ has not formally mapped the transcriptional start site of the T7 promoter, the following reference indicates that the start site occurs at the G following the CACTATA sequence found in the promoter: Nucleic Acids Research, Vol.20, No. 20, pp 4626-4634.
Answer Id: 4435
Will carbenicillin in place of ampicillin help to increase expression levels from the pET TOPO® vectors?
For most purposes, ampicillin works well for selection of transformants and expression experiments. However, degradation of ampicillin is enhanced during the late stages of bacterial growth, when the pH of the culture tends to decrease. This could result in non-selective conditions, resulting in the appearance of satellite colonies on plates, and if the transferred plasmid is unstable it may result in the loss of the plasmid and low expression levels. Carbenicillin is generally more stable under acidic conditions than ampicillin and studies have shown that using carbenicillin in place of ampicillin may help increase expression levels by preventing the loss of the pET TOPO® plasmid. Use at a concentration of 50 ug/ml.
Answer Id: 3883
Does Life Technologies have a vector for co-expression of two proteins in E. coli?
A dual promoter vector is the best option for expressing two proteins at the same time. Unfortunately, we do not offer any prokaryotic expression vectors containing dual promoters. Another option is to use two different but compatible vectors at the same time. For example, you can try using a pET vector such as pET100/D-TOPO® with a pRSET vector. Our pET vectors have a pBR322 origin and our pRSET vectors have a pUC origin, so they are able to replicate in E. coli at the same time. The only issue here is that pRSET is a high copy number vector and our pET vectors are not. Therefore, you may get significantly more protein expression from pRSET in comparison to pET if they are expressed in the same host.
Answer Id: 4181
What PCR enzyme would you recommend for use with the Directional TOPO® Cloning Kits?
For the Directional TOPO® Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50™ or Accuprime™ Pfx and Accuprime™ Pfx Supermix from Life Technologies are recommended for use.
When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO® reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.
Answer Id: 4290
Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?
A. TA Cloning:
- This cloning method was designed for pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 of 15:1 ratio of Taq to proofreader polymerase will still generate enough 3 A overhangs for TA cloning
- The recommended Life Technologies® polymerases include Platinum® Taq, AccuPrime™ Taq, Platinum® or AccuPrime™ Taq High Fidelity, AmpliTaq®, AmpliTaq Gold®, or AmpliTaq Gold® 360.
B. Blunt cloning:
- Use proofreading enzymes such as Pfx50, Platinum® or AccuPrime™ Pfx.
C. Directional TOPO cloning:
- Pfx50 or AccuPrime Pfx work well.
Answer Id: 6651
What are the requirements for primer design when using directional TOPO® cloning?
Please consider the following when designing your primers:
- The 3 pcr primer cannot contain homology to the 5 flap sequence GTGG.
- The enzyme you use must create a blunt-ended PCR product for cloning.
- Primers cannot contain 5 phosphates, which will block the 5 OH nucleophile reactive group.
- The reading frame must be considered when you are designing your primers.
Answer Id: 6738
Can I use a Taq polymerase to generate my gene of interest for directional TOPO® cloning?