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Champion™ pET160 Directional TOPO™ Expression Kit with Lumio™ Technology - FAQs

View additional product information for Champion™ pET160 Directional TOPO™ Expression Kit with Lumio™ Technology - FAQs (K16001)

8 product FAQs found

Can fluorescent protein-expressing cells be fixed?

Yes, fluorescent protein-expressing cells can be fixed using 4% paraformaldehyde in PBS for 10 min followed by one quick PBS rinse and 3 x 5 min washes with 1 mL PBS.

Are the fluorescent proteins offered by Thermo Fisher Scientific (EmGFP, YFP, CFP, BFP and Cycle 3 GFP) humanized?

Yes, all of the fluorescent proteins offered by (EmGFP, YFP, CFP, BFP and Cycle 3 GFP) have been humanized for optimal mammalian expression.

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.

Does Thermo Fisher Scientific 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.

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the source of the EM7 promoter?

The EM7 promoter is a synthetic promoter derived from the T7 promoter. The promoter is typically used to drive expression of antibiotic resistance genes for selection in E. coli.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What protein yields should I expect to get with the pRSET expression vectors?

We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Is the LMG194 sold by Thermo Fisher Scientific the same as that in the ATCC bank?

Yes, it is the same strain. ATCC Number: 47090

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.