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
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
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
I found competent cell vials in my freezer with no box - how can I tell what strain/product it is?
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: 3345
Are your E. coli strains derived from K12?
Is the growth rate of TOP10 cells affected when harboring pZErO®-1 plasmids?
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: 3356
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?
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: 3359
Are TOP10 cells lacIq+ (plus) or lacIq- (minus)? That is, do they produce the lambda lacIq repressor protein?
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: 3361
What is the reason for the nupG mutation in the genotypes for TOP10- and DH10B™-related E. coli strains?
nupG is a mutation for the transport of nucleosides. The nupG site is next to endA on the chromosome, and when endA was mutated by transposon insertion, the nupG site was unintentionally mutated as well. There are no apparent effects of this mutation on cell function or growth.
References: 1) Nghiem, Y. et al. PNAS 85: 2709-2713. 2) Westh Hansen, S.V. et al. Eur. J. Biochem. 168: 385-391.
Answer Id: 3362