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

Does the methylation status of DNA affect its ability to be cloned?

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Answer

Yes. Bacterial host cells will often degrade incoming DNA that has a methylation pattern that is "foreign" relative to that of the cell. Several host strains have been modified to accept mammalian methylation patterns. The modified markers include mcrA, mcrBC, and mrr. Also, endogenous (b-type) restriction endonucleases can be problematic. Modifications of the host to be rK- or rB- are necessary and include hsdR17(AK-, MK+), hsdR17(rK-, mK-), hsdS20(rB-, rB-) or hsdRMS. Strains with the hsdR17(rK-, mK+) mutation lack K-type restriction endonuclease, but contain K-type methylase. DNA prepared from hosts that are rK- mK- is unmethylated and will transform with lower efficiency in rK+ hosts.

TOP10, DH10B™, and OmniMAX™2-T1 cells contain the mcr, mrr, and hsdRMS mutations. Mach1 and standard DH5α™ strains only have the hsdR17(rK- mK+) mutation and are not recommended for cloning eukaryotic genomic DNA.

Answer Id: E3102

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378a592620f9aa1fdb1d7f40611ba6e9_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

How can AmpliTaq® DNA Polymerase be inactivated after PCR?

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Answer

There are several approaches that can be taken to inactivate the AmpliTaq® DNA Polymerase after PCR.

(1) Because AmpliTaq® DNA Polymerase is thermostable, it is necessary to heat it to high temperatures in order for it to be inactivated. Typically, a 99-100 degrees C for 10 min is sufficient.

(2) Raising the EDTA concentration to 10 mM will chelate any free Mg2+. Mg2+ is necessary for enzyme activity. By removing the Mg2+ the enzyme will no longer exhibit enzyme activity.

(3) Phenol-chloroform extraction of the PCR product and ethanol precipitation will also inactivate AmpliTaq® DNA Polymerase.

Answer Id: E1319

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c7919624bf79c38d54c892bfa5bca4c3_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

What advantages do your Stbl2™ cells offer over other cloning strains?

Product FAQ

Answer

There are other strains available that may function similarly to Stbl2™ cells in stabilizing inserts or vectors with repeated DNA sequences. However, one advantage of Stbl2™ cells over many similar strains is that they are sensitive to Kanamycin, so you can use Stbl2 to propagate plasmids containing a Kanamycin resistance marker. 

Answer Id: E4289

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6364f9f083af16fa7dcee833739c00b2_FAQ

I found competent cell vials in my freezer with no box - how can I tell what strain/product it is?

Product FAQ

Answer

Almost all Invitrogen™ competent cell vials are labeled by a laser with the strain name and a batch number. The label is etched into the plastic on the side of the vial, but it may be obscured from view by frost in the freezer.

The cap color can also be used to distinguish between products. Below is a list of cap colors for some of our products.

Chemically competent cells cap colors:
TOP10 One Shot® - Purple; TOP10F' One Shot® - Blue; One Shot® Mach1™ T1 Phage Resistant - Blue; One Shot® OmniMAX™2 T1 Phage Resistant - Pink; MAX Efficiency™ DH5α™ - Brown; Library Efficiency® DH5α™ - Blue; Subcloning Efficiency™ DH5α™ - Clear; One Shot® MAX Efficiency DH5α-T1™ Phage Resistant - Yellow; One Shot® DH10B™ T1 Phage Resistant - Green; INVαF' One Shot® - Clear; MAX Efficiency™ Stbl2™ - Green; One Shot® Stbl3™ - Clear; INV110 One Shot® - Red; BL21 Star™(DE3) - Red; BL21 Star™(DE3)pLysS - Blue; BL21-AI™ - Orange; BL21(DE3)pLysE - Pink; BL21(DE3)pLysS - Green; BL21(DE3) - Brown

Electrocompetent cells cap colors:
TOP10 Electrocomp™ - Yellow; TOP10F' Electrocomp™ - Green; ElectroMAX™ DH10B™ - Yellow; ElectroMAX™ DH10B™ T1 Phage Resistant - Orange; ElectroMAX™ DH5α-E™ - Red; ElectroMAX™ Stbl4™ - Clear

Answer Id: E3345

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768355a64aa44ede9ba9a343949921de_FAQ

Are your E. coli strains derived from K12?

Product FAQ

Answer

Most of our E. coli strains are K12-derived. The exceptions are the BL21 strains (derived from E. coli B), Mach1™ (derived from E. coli W), and HB101. HB101 is derived from a K12/E. coli B hybrid - See FOCUS, 11:3, p. 56.

Answer Id: E3346

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1b03feb45062d109816cb3b00056e554_FAQ

Is the growth rate of TOP10 cells affected when harboring pZErO®-1 plasmids?

Product FAQ

Answer

Yes, the growth rate of TOP10 cells harboring pZErO®-1 will be altered, and depends on how much functional ccdB protein is present. An insert fragment that does not completely disrupt the expression of the LacZ/ccdB fusion (usually very small inserts) will allow some production of the lethal protein which in turn will reduce the growth rate of the cell and produce lower plasmid yields. In contrast, a completely disrupted LacZ/ccdB fusion will allow normal (pUC level) growth and plasmid yield. Typical plasmid yields of pZErO®-1 in LB/Zeocin media are 25% - 75% of pUC grown in LB/Amp. Colonies grown in SOB/Zeocin are more healthy and users can expect 75% - 200% of plasmid yield when compared to pUC grown in LB/Amp.

Answer Id: E3356

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838b175bff4763ab697931f75d01bfd1_FAQ

Why is Beta-mercaptoethanol (BME) no longer included with the One Shot® Chemically Competent E. coli kits? What was the purpose of the BME during E. coli transformation?

Product FAQ

Answer

Beta-mercaptoethanol (BME) degrades carbohydrates on the cell surface, which theoretically allows DNA to get closer to the membrane prior to heat shock. In the past, this was thought to improve the efficiency of transforming E. coli strains, and the addition of Beta-mercaptoethanol was a standard practice for all chemical transformations. However, galU and galK minus strains, such as TOP10, INVαF', DH5α™, DH10B™ and TOP10F', have fewer carbohydrates on the cell surface. After repeated testing of all of our strains, we determined that adding BME had no beneficial effect on transformation efficiency, and we chose to remove BME from the chemically competent One Shot® kits.

Answer Id: E3359

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2cacf394c64d587bff407ea47b9fd5e9_FAQ

Are TOP10 cells lacIq+ (plus) or lacIq- (minus)? That is, do they produce the lambda lacIq repressor protein?

Product FAQ

Answer

TOP10 cells are lacIq- (minus). They do not have the lacIq gene and therefore do not produce the lacIq repressor protein. lacIq is most commonly found on an F' episome, and therefore is present in TOP10F', JM101, JM109, and NM522 strains.

Answer Id: E3361

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28c29a6e6e82ee4e9b0e537847200078_FAQ