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

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Answer

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.

Answer Id: E3259

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6d668e5b96b629314469c673ebbc33f1_FAQ

What is the source of the EM7 promoter?

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Answer

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.

Answer Id: E3270

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f0b4e94eac48c91516111407482622af_FAQ

Will carbenicillin in place of ampicillin help to increase expression levels from the pET TOPO® vectors?

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Answer

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: E3883

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9bcab39babadd031d69eaa69b8afda51_FAQ

Where is the transcriptional start site of the T7 promoter featured in many of Invitrogen™'s vectors?

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Answer

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: E4435

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dc73c49ce03192d2f93e6d0a7e333e45_FAQ

Does Life Technologies have a vector for co-expression of two proteins in E. coli?

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Answer

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: E4181

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4f5919d52c6249bc1a78810d0a3b7107_FAQ

What PCR enzyme would you recommend for use with the Directional TOPO® Cloning Kits?

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Answer

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: E4290

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337cdf091765fed4a7afa0de58e0d347_FAQ

Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

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Answer

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: E6651

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c282cdd6154484d04bb8b2ce3e2275db_FAQ

What are the requirements for primer design when using directional TOPO® cloning?

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Answer

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: E6738

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c1164b31f0912756d36aac7f43b4c2b4_FAQ

Can I use a Taq polymerase to generate my gene of interest for directional TOPO® cloning?

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Answer

No, your gene of interest must be amplified with a proofreading polymerase such as AccuPrime™ Pfx or Platinum® Pfx that leaves blunt ends for directional TOPO® cloning.

Answer Id: E6743

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2b7a1804c053424ca3c174ecc182a20f_FAQ

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

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Answer

You may have to try different ratios ranging from 1:1 to 15:1 insert:vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

Answer Id: E7645

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7f0ffc8664d35f9b979c64a966bd3453_FAQ

What is the best molar ratio of PCR product:vector to use for TOPO® TA cloning? Is there an equation to calculate the quantity to use?

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Answer

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO® cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

Answer Id: E7648

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42f13cc90bca8db8f0cdcf99d3966301_FAQ