I need to buy media to prepare agar plates, which I will be using to grow colonies after transformation (molecular cloning). Which product would you recommend?
We offer two options:
The first is our formulation of LB agar (catalog number 22700-025), which has LB Media and agar mixed together. This comes in a 500g and 2.5 kg size. All you have to do is measure out, add water, and autoclave.
The second option is even easier. Our imMedia™ comes premeasured in pouches. We have three types: imMedia™ Amp Agar, Kan Agar and Zeo Agar for ampicillin, kanamycin and Zeocin™-containing plates. All you have to do is empty the pouch, add water, heat and pour plates. No autoclaving is needed! Each pouch contains sufficient reagents for 8-10 plates.
imMedia™ Amp Agar, 20 pouches, Q601-20
imMedia™ Kan Agar, 20 pouches, Q611-20
imMedia™ Zeo Agar, 20 pouches, Q621-20
For added convenience, you should also check out our LB medium imMedia™ products: (Each pouch makes 200 mL of media)
imMedia™ Amp Liquid, 20 pouches, Q600-20
imMedia™ Kan Liquid, 20 pouches, Q610-20
imMedia™ Zeo Liquid, 20 pouches, Q620-20
Answer Id: 3039
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
How can unstable or toxic DNA inserts be maintained in bacteria?
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: 3099
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 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
How can I clone a gene that has direct repeats and propagate it without altering the repeat sequences?
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 Life Technologies™ 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 Life Technologies™ website.
Answer Id: 3867
You offer competent cells in Subcloning Efficiency™, Library Efficiency® and MAX Efficiency™. How do these differ?
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: 3869
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
How can I tell whether or not detergent in a sample can be removed with Prosorb® Sample Preparation Cartridges?
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: 1269
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 often should the sound of the vacuum assist be heard on the 433A?
At most, once or twice a day. If more frequent, there may be a gas leak. Nitrogen pressure is used to generate the vacuum, which assists the opening of the valves. If there is no apparent gas leak, then it is possible that a valve has failed and the solvent leakage has damaged the vacuum ballast. Both the vacuum system and the valve block need inspection.
Answer Id: 1315
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
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