When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

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Answer

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Answer Id: E1320

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306a0ad00215a85bf5c8a4fef43bb80d_FAQ

You offer competent cells in Subcloning Efficiency™, Library Efficiency® and MAX Efficiency™. How do these differ?

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Answer

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:
Subcloning Efficiency™ cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency™ cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency™ cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA

Answer Id: E3869

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e5e419aa63dc67b211dedc933627d891_FAQ

How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

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If amplification products are generated in the control tube/well that contains no reverse transcriptase (ie., the no-RT control), it may be necessary to eliminate residual genomic DNA from the RNA sample. Use the following protocol to remove genomic DNA from the total RNA preparation.

Add the following to an autoclaved 0.5 mL microcentrifuge tube on ice:
(1) Total RNA (ideally, less than or equal to 1 μg, see Note 1)
(2) 1.0 μL of 10X DNase buffer (200 mM Tris 8.3, 500 mM KCl, 20 mM MgCl2)
(3) 0.1 U-3.0 U of DNase I (RNase-free, Cat. No. 18047-019) or 1.0 U Amplification Grade Dnase I (Cat. No. 18068-015, see Note 2)
(4) Bring volume up to 10 μL with DEPC-treated water.
(5) Incubate at room temperature for 15 min. See Note 3.
(6) Terminate the reaction by adding 1 μL 25 mM EDTA and heat 10 min at 65 degrees C. See Note 4.
(7) Place on ice for 1 minute.
(8) Collect by brief centrifugation. This mixture can be used directly for reverse transcription.

***NOTE 1: To work with higher quantities of RNA, scale up the entire reaction linearly. Do not exceed 2 μg RNA in the 10 μL reaction. More RNA will increase the viscocity of the solution and prevent the DNAse I from diffusing and finding the DNA.

***NOTE 2: Amplification Grade DNAse I has been extensively purified to remove trace ribonuclease activities commonly associated with other "RNAse-free" enzyme preparations and does not require the addition of placental RNAse inhibitor.

***NOTE 3: It is important not to exceed the 15 minute incubation time or the room temperature incubation. Higher temperatures and longer times could lead to Mg++-dependent hydrolysis of the RNA.

***NOTE 4: This procedure requires careful pipetting of all solutions so that the concentration of divalent metal cation (Mg++) is controlled.
Because the DNAse I must be heated to 65 degrees C to inactivate the enzyme, the concentration of free divalent metal ions must be low enough (less than 1 mM) after addition of the EDTA to prevent chemical hydrolysis of the RNA. See references below.
After the addition of EDTA, there is an approximately 1:1 molar ratio of Mg++ : EDTA. EDTA chelates Mg++ molecules on a 1:1 molar basis. Therefore, this RNA can be directly used in a reverse transcription reaction. First-strand reverse transcription buffers typically result in a final concentration of 2.5 mM Mg++. If the reverse transcription buffer does not contain MgCl2, add it to the reaction at a final concentration of 2.5 mM. This results in a net final concentration of approximately 2.25 to 2.5 mM MgCl2.

RNA hydrolysis references:
Molekulyarnaya Biologiya Vol 21: 1235-1241 (1987).
References on the mechanism of hyrolysis by other cations:
Eichorn, G.L. and Butzov, J. Y. Biopolymers 3:79 (1965)
Butzov, J. Y and Eichorn, G.L. Biopolymers 3:95 (1965)
Farkas, W.R. BBA 155:401 (1968)
The author of the first paper expresses the opinion that the mechanism of the non-specific hydrolysis by cations which proceeds through 2',3' cyclic phosphate formation is similar to that of specific hydrolysis such as RNA splicing.

Answer Id: E4152

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54c668c14d60789a6e642e5fab8dcd39_FAQ

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

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Answer

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

Answer Id: E4159

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764c1acf23753110d170b6570530b472_FAQ

What is the promoter used in the Bac-to-Bac® expression system?

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Answer

The promoter that drives the gene of interest is the polyhedron promoter. This promoter can be substituted by the p10 promoter, though the polyhedron promoter is generally 3-5 times stronger for most proteins. However, a protein that is highly modified or secreted is often expressed much more efficiently by the p10 promoter, as it becomes active in very early late phase, as opposed to the polyhedron promoter, which is not active until very late phase. Cellular protein synthesis that is required for the efficient and correct processing of complex proteins is shut down during the very late phase. This explains why some reports mention the expression of secreted and modified proteins where the p10 promoter is just as efficient as the polyhedron promoter or as much as 2x higher than the polyhedron promoter. In most cases, however, the polyhedron promoter is working just fine. The determination as to which is the stronger promoter will depend on a number of factors, including the nature of the protein and the time of harvest post-infection.

Answer Id: E9407

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59df3454e141483fd107b8ac0862ad70_FAQ

What is the size of the baculovirus polyhedron protein?

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The polyhedron protein is 30 kDa.

Answer Id: E9408

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bdcc115309794350b91e3ee33ae34686_FAQ

I’m interested in expressing a large protein, larger than 130 kDa. Can I use the baculovirus system or will the proteins be degraded?

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Answer

Our R&D team has successfully expressed proteins up to 300 kDa. If they express in >2% serum, it should minimize degradation. If you don’t mind the extra step of purification, 10% serum could be used. We highly recommend doing a time-course infection with high-titer stock, with a MOI of 5-10, to make an assessment of the minimum harvesting time necessary for the best expression. Time points should be taken every 24 hours for 5 days.

Answer Id: E9409

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60a115e266d9da747f91e80fb92de604_FAQ

Are there preferred secretion signals for use with baculovirus?

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Answer

Insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. Please see the following references:

- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754-16762.
- Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177-183.

Answer Id: E9410

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b915b52764126c8d36e4ac86c280b171_FAQ

Is there a packaging limit for the baculovirus?

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Answer

The baculovirus rod will continue to elongate as required to package the DNA. Thus, the system could theoretically accommodate hundreds of Kb. Standard cloning techniques will limit the insert size before packaging limits become an issue.

Answer Id: E9411

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08844716d86c9385eefb9c4b4fb8d3b9_FAQ

What steps are recommended for preventing proteolysis in the baculovirus expression systems?

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Answer

The following is an excellent reference for how to prevent proteolytic artifacts in the baculovirus expression system:
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18-20.

Answer Id: E9412

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4464128c515d6e01a305e76b92faabd1_FAQ

What is the difference between the Bac-N-Blue™ expression system and the Bac-to-Bac® expression system?

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Answer

In the Bac-N-Blue™ system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue™ vector is a linearized AcMNPV derivative that contains an incomplete (3’) lacZ fragment. The corresponding transfer vector contains a 5’ lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue™ baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue™ baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac® expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac® expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 ™μg/mL kanamycin (bacmid), 7 ™μg/mL gentamycin (pFastBac™), 10 ™μg/mL tetracycline (helper plasmid), 100 ™μg/mL Bluo-gal, and 40 ™μg/mL IPTG.

Answer Id: E9419

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0ff3b794c77db2d1e35a8f333733571c_FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac® expression system and all the colonies obtained are white. Shouldn't I expect to see some blue colonies?

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Answer

Although you will be picking white (recombinant) colonies, you should expect to see some blue (contain non-recombinant bacmid) colonies. Here are some possible causes for seeing no blue colonies and recommendations for the same:

- Insufficient time for color development: Wait at least 48 hours before identifying colony phenotypes.
- Use X-Gal instead of Bluo-gal in agar plates: Use Bluo-gal in plates to increase contrast between blue and white colonies.
- Insufficient growth after transposition: Grow transformed cells in SOC medium for a minimum of 4 hours before plating.
- Bluo-gal and IPTG omitted from plates: Prepare fresh selective plates containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal, and 40 μg/mL IPTG.
- There are too many colonies on the plate: Serially dilute the transformation mix to obtain well-spaced colonies (10-2 to 10-4 is suggested).
- Plates are too old or stored in light: Do not use plates that are more than 4 weeks old; store plates protected from light.
- Incubation period too short or temperature is too low: Wait at least 48 hours before picking colonies. Incubate plates at 37 degrees C.

Answer Id: E9438

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eedb8e7ff9298847ebbe32a82cd7a5d4_FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac® expression system and all of my colonies are blue. What could be the cause of this and what should I do?

Product FAQ

Answer

Please review the following possibilities and recommendations:

- pFastBac™ DNA used for transformation was of poor quality: Use purified plasmid DNA for transformation and check the quality of your plasmid DNA.
- Gentamicin omitted from plates: Prepare fresh selective plates containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal, and 40 μg/mL IPTG.

Answer Id: E9439

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ad2a50283c453f6d897da6f1001f3ce0_FAQ

I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac® expression system and I’m getting very few colonies. What could be the cause for this and do you have any recommendations for how to fix this?

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Answer

Please review the following reasons and our recommendations:

- Use LB medium for recovery/expression period: Use SOC medium for the 4 hr growth time.
- Recovery/expression time too short: Increase the recovery time to > 4 hr at 37 degrees C or 6 hr at 30 degrees C.
- IPTG concentration is not optimal: We suggest using 20-40 μg/mL IPTG.

Answer Id: E9440

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c8a367139ac2fa717e63ca14b33a779d_FAQ

I transfected my bacmid DNA but saw no signs of infection. The quality of my DNA prep might be an issue. How can I analyze the bacmid DNA?

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Answer

We recommend running 1/8th of the 40 μL midiprep sample on a 0.5% TAE agarose gel. Electrophorese slowly at 23 V for 12 hr. The banding pattern of the recombinant bacmid midiprep should be seen.

Answer Id: E9441

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20cada4aadb38da0c007341165e59e12_FAQ