Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?
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: 3098
Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?
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: 3100
Can encapsulated phagemid DNA or M13 phage be used to infect bacteria?
Single-stranded DNA viral particles like M13 require the presence of an F pilus in order to infect E. coli. This criterion is met by TOP10F', DH5α F'IQ™, INVαF', Stbl4™, OmniMAX™2-T1 and DH12S™ cells. These cells are not traD mutants, which effectively allows the cells to retain the F' episome. Transforming single-stranded DNA can cause a 100- to 1,000-fold reduction in efficiency compared to viral particles.
Answer Id: 3101
Does the methylation status of DNA affect its ability to be cloned?
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: 3102
Can I re-use my competent cells once the tube has been thawed?
Yes, competent cells can be thawed and re-frozen at least once, but be aware that each freeze-thaw cycle can result in up to a 10-fold reduction in transformation efficiency.
To re-freeze unused competent cells, we recommend the following protocol: Pre-cool some new empty vials on ice for 5 min. Thaw the cells, and then aliquot a single-use volume of cells (usually 20-100 ul as recommended in the product manual) into the new tube. Freeze the cells immediately in a dry ice-ethanol bath. (Be sure that ethanol does not leak inside the tube - keep the level of ethanol well below the cap.) Transfer the frozen cells immediately to a -80C freezer, and do not thaw them again until ready for use.
Answer Id: 3103
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?
How can peptide synthesis amino acid cartridges leak and spill solvents during their activation and transfer to the RV?
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: 1316
What causes synthesized \peptides to be over or under the expected weight when post-cleavage mass spectral analysis is performed? What causes multiple peaks for one peptide, by RP-HPLC?
Overweight: Most often reflects failure to remove all the side-chain protecting groups from all amino acids ("incomplete cleavage"). If insufficient amounts of scavengers were used, these protecting groups may reattach, OR permanently attach to another amino acid. If this occurs, it may be necessary to modify the system used for cleavage. Increased time, better mixing, or increased scavengers may be needed. This may be best determined by trial and error.
Underweight: Usually reflects a problem during the synthesis. If the run was done with conductivity or UV monitoring during Fmoc removal, it may be possible to modify the subsequent synthesis to avoid problems at specific areas of the sequence. An analysis of the probable secondary structure of the peptide chain may also be helpful for future strategies.
Answer Id: 1318
How can AmpliTaq® DNA Polymerase be inactivated after PCR?
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: 1319
When should DMSO, formamide, glycerol and other cosolvents be used in PCR?
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: 1320
What is the formulation of the SOC medium that is provided with competent cells?
SOC (Super Optimal Catabolite) Medium Preparation (for 1 Liter):
1) To a 2 Liter flask with stir bar add the following:
- Bacto Tryptone 20 g
- Yeast Extract 5 g
- Sodium Chloride (NaCl) 0.58 g
- Potassium Chloride (KCl) 0.186 g
2) Add sterile water to a final volume of 1 Liter.
3) Mix well on magnetic stir plate for 5-10 minutes or until all of the ingredients are well mixed and completely dissolved.
4) Autoclave 30 minutes.
5) Allow to cool to room temperature.
6) Add 10 ml of sterile 2M Magnesium Solution (1M Magnesium sulfate, 1M Magnesium chloride)and mix well.
7) Add 10 ml of sterile 2M Glucose and mix well. (Final Glucose concentration is 20 mM).
Answer Id: 3343
Do any Invitrogen™ competent cells contain DMSO in the freezing medium?
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: 3355
How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?
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: 4152
How do you recommend that I prepare my DNA for successful electroporation of E. coli?
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: 4159
What advantages do your Stbl2™ cells offer over other cloning strains?
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: 4289