Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

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Answer

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

Answer Id: E3098

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7b9af43130a405a9d9e93366e5befc2c_FAQ

Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

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Answer

Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

Answer Id: E3100

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7823454388724302e4b98f8e4937b70f_FAQ

How can I tell whether or not detergent in a sample can be removed with Prosorb® Sample Preparation Cartridges?

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Answer

If the detergent identity is known, look up its CMC (critical micellar concentration); a table of these is listed in the Biological Detergents section of the Sigma-Aldrich® catalog. If you are above the CMC, the detergent forms micelles that cannot be removed by the filtering membrane, so the sample must be diluted prior to application to the Prosorb® Sample Preparation Cartridges.

Answer Id: E1269

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29547cb90ed15729eb5ffeec9c1b3cac_FAQ

Why doesn't the 433A manual or the "quick start card" mention the need for an extra AA cartridge in the guideway at the start of a sequence?

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Answer

The newest 433A User's Manual does cover this. The barcode reader is one position ahead (left) of the needle position. The extra (empty) is needed to prevent advancement of the first cartridge until after it is read.

Answer Id: E1314

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3f1503354f2db909967f808daddcfab3_FAQ

How can peptide synthesis amino acid cartridges leak and spill solvents during their activation and transfer to the RV?

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Answer

If these cartridges are being reused, the NMP can cause them to swell, and they no longer fit or slide well in the guideway. If the guideway or the exterior of the needles have became dirty, this can also lead to misalignment. And if you forgot to remove the metal cap, the needle cannot penetrate the septum - this may cause a spill OR stop the run.

Answer Id: E1316

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2f0fb91467ba67924b591da3bec23914_FAQ

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

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

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Answer

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

Do any Invitrogen™ competent cells contain DMSO in the freezing medium?

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Answer

Yes, several of our competent cells products are frozen with DMSO. The presence of DMSO (dimethylsulfoxide) will generally be indicated in the MSDS files if you have a question about a particular product, but here is a list of commonly used products that are known to have DMSO in the freezing buffer:

One Shot® OmniMAX™ 2 T1 Phage Resistant Cells, Cat. No. C8540-03

One Shot® INVαF' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot® MAX Efficiency® DH5α-T1™ Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency® DH5α-T1™ Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency® DH5α™ Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency® DH5α™ Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency® DH5α F'IQ™ Cells, Cat. No. 18288-019

MAX Efficiency® Stbl2™Chemically Competent Cells, Cat. No. 10268-019

Answer Id: E3355

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828d82fb12e1ee28fd90e20019baaf48_FAQ

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

Product FAQ

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

I am trying to clone an insert that is supposedly pretty toxic. I used DH5α and TOP10 cells for the transformation and got no colonies on the plate. Do you have any suggestions for me?

Product FAQ

Answer

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

- After transforming TOP10 or DH5α cells, incubate at 25-30°C instead of 37°C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
- Try using TOP10F' cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
- Try using Stbl2 cells for the transformation.

Answer Id: E7646

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3cc4791dca6824d082228a9a9bc66c9f_FAQ