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

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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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How can unstable or toxic DNA inserts be maintained in bacteria?

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There are a few steps you can take to improve stability of clones with difficult-to-maintain inserts. Supplement the medium with extra nutrients (e.g., add 20-30 mM glucose to Terrific Broth) or try a vector that has a reduced copy number (e.g., pBR322). Some clones can exhibit a high degree of deletions; this is usually a result of the clones having long terminal repeat (LTR) sequences or regions with high secondary structure. To overcome this problem, the cells can be grown at 30°C or ambient temperature (in LB or in a nutrient rich broth like Terrific Broth). Do not to let the cells reach late stationary phase in liquid culture. Alternatively, transform into cells that maintain unstable sequences such as Stbl2™, Stbl3™, or Stbl4™ cells.

Answer Id: E3099

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I am trying to clone an insert that is supposedly pretty toxic. I used DH5? and TOP10 cells for the transformation and got no colonies on the plate. Do you have any suggestions for me?

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Answer

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

- After transforming TOP10 or DH5? 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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Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

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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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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™ INV?F' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot™ MAX Efficiency™ DH5?-T1™ Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency™ DH5?-T1™ Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency™ DH5?™ Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency™ DH5?™ Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency™ DH5? F'IQ™ Cells, Cat. No. 18288-019

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

Answer Id: E3355

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

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

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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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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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How can I clone a gene that has direct repeats and propagate it without altering the repeat sequences?

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Answer

The first thing you can do is to lower the growth temperature of your E. coli cells when propagating your plasmid containing the unstable gene. Slowing the growth of any cell strain at 30C, 25C or even lower can help to stabilize the replication of the plasmids they contain.

If your sequence is still unstable despite low-temperature growth, there are also specific bacterial strains available that can further help to stabilize repeated sequences during propagation. Invitrogen™ Stbl2™ and Stbl4™ competent cells are both designed to improve stability when cloning retroviral or direct repeat sequences.

In a series of experiments, Stbl2™ was compared directly to several other strains also known for increasing stability of retroviral and tandem repeat inserts. An article in the Focus Journal (Issue 16.3, p. 78) contains data from two such experiments – the full article can be found on the Thermo Fisher Scientific website. A brief summary of the data is included below:

Stability of clones containing SIV retroviral sequences:
Stbl2™ @ 30°C - 100%; Stbl2™ @ 37°C - 100%; HB101 @ 30°C - 100%; HB101 @ 37°C - 100%; SURE @ 30°C - 72%; SURE @ 37°C - 0%

Stability of clones containing 100 repeats of a 32-bp sequence:
Stbl2™ @ 30°C - 89%; Stbl2™ @ 37°C - 73%; HB101 @ 30°C - 15%; HB101 @ 37°C - 0%; SURE @ 30°C - 53%; SURE @ 37°C - 0%

Results from a separate experiment on stability of a tandem repeat of four R67 dihydrofolate reductase genes in Stbl2™ vs. SURE cells can be found in Focus 19.2, p. 24 on the Thermo Fisher Scientific website.

Answer Id: E3867

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