FAQ
ITS-M
Stock ITS-M is 500X and working concentration is 1X. Please check working concentration of ITS-M first.
If working concentration of ITS-M is correct. Slow growth might due to adaption of new culture supplement, especially for adhesion cell line. According to our experiments, it might take 2 weeks to come back to normal doubling time.
In addition, we suggest to adjust ratio of serum and ITS-M progressively (gradually reduce % of FBS) for cells adapting to new growth environment.
Due to cholesterol will be precipitated after storing at 4°C.
We suggest to centrifuge tube by 12,000g for 3 minutes and aliquot from supernatant.
NAP-RNA
Possible causes are as below. First of all, the sample was not fresh, thus causing the cells to die and the RNA degradation. Further, the operation steps were not executed solidly; the sample was not fully lysed. Last, the elution buffer must use the RNase-free H2O. When doing the elution, place in the oven at 60°C for 10 minutes when performing elution.
Please use DNase A to remove DNA.
It indicated the residual presence of alcohol. Place the column in the oven at 60°C for 10 minutes.
The sample was added excessively, causing the lysis step to be executed incompletely. When sucking the supernatant, avoid taking in the impurities.
It indicated the presence of RNase in the working environment. Please use the clean instruments or the RNase inhibitor.
NAP-Genomic DNA
Buffer B with the incorrect ratio was added to the amplification reaction.
Verify that an equal volume of the Buffer B was added to the reaction.
The ethanol(96~100%) was not added to the W2 buffer.
The gel slice did not dissolve completely.
The gel slice was too big. If the column was overloaded, decrease the loading volume. If overloaded , separate into 2 columns. If the DNA fragments were more than 300mg, separate the gel slice into two microcentrifuge tubes.
Dissolved incompletely: Increase time for the Gel Extraction Step until the gel slice has completely dissolved. Use an equal volume of the Buffer B and/ or low-melting-point agarose gels.Incorrect elution conditions: Ensure that the Buffer E or ddH2O is added into the center of the PG Column.
The recovery buffer volume was too small. Increase the amount of the Buffer E to at least 50 μl for use.
Please use RNase A to remove RNA.
It indicated the residual presence of alcohol. Place the column in the oven at 60°C for 10 minutes.
The sample was added excessively, causing the lysis step to be executed incompletely. When sucking the supernatant, avoid taking in the impurities.
NAP-PCR and Gel
Buffer B with the incorrect ratio was added to the amplification reaction. Verify that an equal volume of the Buffer B was added to the reaction.
The ethanol(96~100%) was not added to the W2 buffer. The gel slice did not dissolve completely.
The gel slice was too big. If the column was overloaded, decrease the loading volume. If overloaded , separate into 2 columns. If the DNA fragments were more than 300mg, separate the gel slice into two microcentrifuge tubes.
Dissolved incompletely: Increase time for the Gel Extraction Step until the gel slice has completely dissolved. Use an equal volume of the Buffer B and/ or low-melting-point agarose gels.Incorrect elution conditions: Ensure that the Buffer E or ddH2O is added into the center of the PG Column.
The recovery buffer volume was too small. Increase the amount of the Buffer E to at least 50 μl for use.
Use the ethanol to precipitate the DNA, or repurify the DNA fragments and elute with the nuclease-free water.
Remove the EtOH in the hood briefly.
Following the Wash Step, dry the PG Column with additional centrifugation at 14~16,000 x g for 2 minutes.
Check the loading volume. If overloaded , separate into two columns. Ensure that any buffer prepared in the laboratory was prepared according to the instructions.
Make sure that no residual ethanol remains in the membrane before eluting the plasmid DNA. Re-centrifuge if necessary.
NAP-Plasmid DNA
Please confirm and follow the manual’s instructions accordingly. There would not be a sufficient amount of plasmid if the lysis step was not performed thoroughly.
Elution buffer<pH7.0, it was unable to confirm the eluted plasmid.
If the cultured bacterial species exhibited a low copy number of plasmid, the bacterial culture volume needed to be increased. Plasmid lost in E.coli, please re-culture with the freshly prepared bacteria. Elution buffer, pre-heat at 70°C.
Please make sure RNase A was added into the Buffer M1 and stored at 4°C.
It indicated the residual presence of alcohol. After adding the wash Buffer2 , re-centrifuge for the Centrifuge Step or place the column in the oven at 60°C for 10 minutes. For the RNA or DNA interference, remove and clean it well.
It indicated plasmid DNA degradation. Please shake it carefully when performing it. Keep plasmid preparations on ice.
Do not overgrow bacterial cultures. Do not incubate more than 5 minutes after adding the Buffer M1.
Nimble Juice
There are few reasons.
1) Inappropriate detection method:
The gel stained with Nimble Juice might be exposure under the UV light, and be sure the wavelength of excitation around at 330 / 390 nm, and the wavelength of maximum at 570 nm.
2) Long washing condition: we suggest to shortly washing gel by 60 ml ultra pure water for 1-5 minutes after staining.
There are few possibilities.
1) Inappropriate stain procedure: Operating Nimbe Juice Series will be used microwave oven. We suggest to put the box lid on to reduce water evaporation when doing heating to avoid dry buffer caused.
(Please use heat insulator to take box out.)
2) Over staining.
More detail, please refer to package insert.
The prestained protein ladder contains the blue-color dye, which might have the negative staining after Nimble Juice staining. We suggest to change the unstained protein ladder on your experiment.
Yes, but Nimble Juice will influence following experiments. We suggest to immerse gel in ultra pure water or general saline buffer for few hours before next experiment.
Protein Ladder
Most of the common gel running buffers are composed of Tris-glycine or Tris-tricine. Tris-glycine buffer systems are useful for the separation of proteins over a wide range of molecular weights (5-300 kDa) and are compatible with denaturing or non-denaturing conditions. The Tris-tricine buffer is generally recommended for the electrophoresis of low molecular weight proteins and peptides (<10 kDa) that need to be reduced and denatured prior to loading. The Tris-acetate buffer system is used for the separation of larger proteins (>200 kDa).
Protein ladder bands can sometimes be detected with chemiluminescent techniques due to non-specific interactions of ladder proteins with either primary or secondary antibodies (or with both). The ladder bands are only rarely detected by chromogenic substrates. The extremely high sensitivity of the chemiluminescent assays is needed to see the bands, so the actual degree of cross-reactivity is low. The non-specific cross-reactivity is difficult to predict as it often has a different pattern depending on the antibodies used. If antibodies recognize a linear epitope, the cross-reactivity may be due to sequence homology. If antibodies react with a denaturation-resistant conformational epitope, it could be impossible to identify the exact reason for the detected cross-reactivity. The most general way to handle this problem would be to use lower concentrations of antibodies.
We do not provide precise protein concentrations in the Certificate of Analysis. We recommend that researchers should not do in-gel quantification; they should instead perform a BSA dilution series. If a researcher insists on doing in-gel quantification, it must be done with an unstained ladder.
When a protein is covalently bound to a charge-carrying dye molecule, this can affect the protein’s overall charge.
Altering the protein’s charge will most likely change its mobility within the gel. This explains why the prestained protein ladders are given “apparent” molecular weight values, while regular unstained protein ladders are given their true molecular weights. The apparent molecular weights stated on the data cards and other references of our prestained protein ladders were determined using Tris-Glycine, Tris-tricine, or Bis-Tris gels. The apparent molecular weight may seem “incorrect”. The reason for this disparity is the different formulations of the gel types (buffering agents, ionic strength, and pH).
Most of time it is not happened. However, few strong detergent might cause red and green color reduction due to relative lower binding strength of these two colors by washing with strong detergent several times.
As following, we suggest the formulation of wash buffer and the stripping buffer:
Wash buffer
10mM phosphate buffer, pH 7.4, 2.7mM KCl,
140 mM NaCl,
0.05% Tween 20
Stirpping buffer
0.2 M Glycine, pH2.8,
0.5M NaCl
Every batch of protein marker has passed strict QC before shipping. Degradation might be caused by unappreciated storage condition. We suggest to aliquot PM to avoid frequent freeze-thaw and store at -20°C. Another major possibility is contaminated by protease in environment. We suggest you to sanitize by ethanol before starting experiment, ware glove and do not reuse tips.