- Q:How do you check the quality of the RNA on your frozen tissue sections? Do you perform in situ hybridization tests for each of your blocks?
A: BioChain doesn't isolate total RNA from frozen tissue sections. Instead, total RNA is isolated from frozen tissues prior to sectioning. In order to ensure quality, at least one of the tissue slides from each lot is stained with H & E.
- Q: Do you usually use OCT embedded tissue block as the starting material or frozen tissue block without OCT to make frozen tissue sections?
A: We usually use OCT embedded tissue block as the starting material to make frozen tissue sections.
- Q: I saw your frozen section was fixed by aceton. Can I get frozen section without fixation?
A: Yes, you can if you mention this request specifically.
Genomic DNA Extraction Kit
- Q: When isolating DNA from a plant using a genomic DNA Extraction Kit, how much starting tissue is needed and what is the yield?
- A: If you are extracting from one gram of plant tissue, it is estimated that about 100ug - 200ug of genomic DNA can be extracted
- Q: Can the Genomic DNA Extration Kit be used on plants?
- A: Yes, the Genomic DNA Extraction Kit can be used on plants.
- Q: Can the Genomic DNA Extraction Kit be used to isolate DNA from paraffin embedded tissues?
- A: Yes, but you need to deparafinize the tissue section first. Also, the quality of the DNA is very poor. (100-1000 times poorer than from fresh tissue)
- Q: What is the minimum amount of tissues and cells needed to isolate total RNA using our extraction kit?
- A: Using the Total RNA Isolation Kit to extract RNA leukocy from 50ml will yield a maximum of 500 ug, which is = to 10 ug per 1 ml.
- Q: Is Biochain's Genomic DNA Extraction Kit suitable for extracting from Alcaligenes Eutrophus?
- A: Theoretically, our genomic DNA extraction kit is ideal for any kind of bacteria genomic DNA isolation. Although we do not have experience performing the isolation from Alcaligenes Eutrophus
- Q: Is the genomic DNA extraction kit suitable for use on a mosquito?
- A: It should be but it is not tested.
- Q: Can BioChain's genomic DNA isolation kit be used on insects and earth worms?
- A: Yes
- Q: What is the minimum sample required, per reaction of blood cells, tumor cells and tissues for this kit?
- A: You can use 0.1 grams of blood or tumor cells and reduce the reagent amount proportionally. For tissues, it depends. Example: For high yield tissues such as liver, you may start from 0.05g and reduce the reagent amount proportionally. But for low yield tissue, such as adipose, you may start from at least 0.5.
- Q: I am interested in the miRNA product: miR-24 One-Step qRT-PCR Detection Kit. Do you have anything published about this product?
- A: Thank you for your interest. We just recently launched this product. Currently we do not have anything published. The kit's manual can be downloaded from the website.
Paraffin Tissue Section & Array
Q: What's your recommendation for preventing paraffin sections from falling off the slides?
A: Avoid using any solutions with detergents such as Triton X100, or Tween 20. These detergents may cause the tissue to fall off the slides.
Q: Is the paraffin tissue section suitable for RNA in situ hybridization?
Q: How did you treat the tissue to make paraffin tissue section?
A: The tissue was fixed in formalin immediately after harvest, then dehydrate and embedded in paraffin.
Q: What have the sections been coated with? for example, do you use 3-aminopropyltriethoxysilane?
A: The sections have not been coated with any thing.
Q: How long were tissue fixed after harvest from the body, either through biopsy or autopsy?
A: 24-48 hours.
Q: What did you use to fix the tissue?
Q: Do cerebellum tissue sections contain the following structures: external granular layer, internal granular layer, molecular layer, purkinje layer and deep cerebellar nuclei?
A: Yes, they do.
Q: Are your paraffin arrays applicable for FISH? Is it fixed homogenously on the slide?
A: When we claim the tissue array is suitable for ISH, that means it fits FISH as well. All the tissues are fixed homogenously.
Q: How many spots are on each of you tissue array? What is the size of each spot?
A: There are 24 to 150 spots in our tissue arrays. Most of the spot size is 1.5 mm diameter.
Q: Was the heart which is used to make tissue section perfused?
A: No, it was not.
Q: Is it possible to have the genomic DNA made up in PCR grade water rather than TE buffer?
A: Yes, but there will be an additional charge because we need to precipitate the DNA first and redissolve it in PCR grade water.
Q: What is the concentration of your Genomic DNA?
A: The concentration may vary from lot to lot, but in general, the concentration for is between .5-3 ug/ul.
Q: Are all your cDNAs from total RNAs that have gone through DNase I digestion?
Q: Can the optimax cDNA synthesis kit be used as direct tissue to cDNA synthesis or direct cell to cDNA synthesis?
A: No. It has to be started from total RNA or mRNA.
Q: What is the difference between the cDNA Library (Normarlized) and the other cDNA libraries that BioChain sells?
A: The molecular complexity of a cell, makes analysis of primary cDNA libraries problematic, especially if sequence determination is being applied for gene discovery efforts. Normalization is a complex molecular process where the cDNA copies of a primary library are equalized so that each original mRNA transcript is represented in the normalized library to the same extent as all the rest of the clones. This means that the number of copies of the high and moderately abundant genes are reduced in the library to the levels of the rare genes. Now, with a normalized cDNA library, sequence analysis does not repeatedly pick up the same gene and the rare transcripts become more accessible.
Q: For cDNA, do you provide the same beta-actin control primer for all species?
A: Yes, our beta-actin primer control can be used for all our current animal species.
Q: I need to isolate open reading frames for a small group of cDNAs. Would it be possible to do so by PCR amplification of your library or would it be necessary to screen the library using colony blots?
A: If the target gene's sequence is known, then it will be easier to design the primers to "fish" out the target gene by PCR. It's not necessary for you to screen the library by colony blots. These days it seems that fewer people are doing screening library work unless their target gene is unknown, and they only have a partial sequence of the target gene, and they have to use it as a probe to screen the library.
Q: What quality control method do you use to make sure your total RNA is good? Do you run bioanalyzer or northern?
A: The integrity of the RNA is examined by visual inspection for the presence of intact bands of 18s and 28s ribosomal RNA when electrophoreses on a denaturing agarose gel. The quality and purity of total RNA were tested by spectrophotometer. A260/280 is between 1.8 and 2.0 (detected in 10 mM Tris-Cl, pH 7.5). The RNA is treated by DNase I, and is tested as DNA free RNA by PCR. However, please be advised that total RNA from some tissues may not be treated by DNase I. Biochain is not using Agilent Bioanalyzer as a standard QC method for every total RNA products, though 80% of standard total RNA products are with Agilent Bioanalyzer data. If customers request RNAs without Agilent bioanalyzer data to be tested on Agilent Bioanalyzer, additional fee will be applied. To visualize the RNA images on agarose gel, we recommend the same gel system be used as Biochain?span lang="zh-cn">'s (1% agarose gel in 1xMOPS buffer with formaldehyde). Biochain is not responsible for customer?span lang="zh-cn">'s getting degraded RNA images from other gel systems, such as TAE gel, TBE gel, Urea gel, etc.
Q: If I can't get any product from the cDNA OR RNA, do I get another lot of samples?
A: Yes, we can provide a different lot as a replacement, however only for special cases. One such case is if our control primer doesn't work. This is very rare. We need to look at the case deeply before we agree to send a different lot. We don't and can't guarantee that you will be able to amplify your gene of interest. In fact, I don't know of any other company who can. The probability of you successfully amplifying your gene lies on several factors. First factor is whether or not your gene of interest is high in abundance. In other words, is your gene scientifically known to be highly expressed in the tissues of your interest? Please be advised that all of our cDNAs were reverse transcribed from total RNA and not mRNA. If they are very low abundant genes, then I will suggest you get some mRNA first, and use the mRNA as template to synthesis cDNA, this will increase the chance to clone low abundant genes. Several of my customers were successful in cloning low abundant gene by using mRNAs. However, please keep in mind that we don't and can't guarantee that you will be able to amplify your gene in the end. By starting from mRNA it just increases your chances of amplification but doesn't guarantee amplifictaion. Second factor is if your target gene's GC content is high. If the GC content is high, then you should add either Betaine or DMSO in your PCR reaction, this will help you a great deal in amplifying the gene. Third, if you want to amplify a very big size, for example, over 6 kb, it will be difficult.
Q: What is the concentration of your cDNA?
A: Our cDNA is PCR ready cDNA, 1 ul is good for 1 PCR reaction, since 11 ug Total RNA was used to produce 40 ul cDNAs, the concentration of the cDNA should be about 2.5 ng/ul.
Q: I used your cDNA as template to amplify my target gene, but I can not get the signal, while I did get good signal of a house keep gene, what is the problem?
A: It is possible that the gene of your interest is a GC rich gene, optimizing your PCR condition by adding 0.5 M Betaine may help you solve the problem. Also, your target gene may be a low abundant gene, in such case, we would recommend you purchase mRNA either from BioChain or other sources, and use mRNA as template for cDNA synthesis.
Q: If I use the same amount of cDNA for different volume of PCR reaction, will I get different amount or concentration of PCR products?
A: Theoretically, the PCR product amount should be the same amount but the concentration should vary depend on the PCR volume. If a very faint band from the first round PCR was obtained, then a sharper band should be obtained with a second round PCR using the first round PCR product as templates.
Q: Can your total RNA extraction kit be used to extract RNA from leukocyte and bone marrow? What about your Dr. P Kit?
A: Yes for both.
Q: Can the total RNA extraction kit be used on plants?
A: Yes, total the RNA extraction kit can be used on plants.
Q: If isolating from plants using the total RNA extraction kit, how much starting tissues does the customer need and what would be the yield?
A: If extracting from 1 gram of plant tissue, it's estimated that about 600 ug of Total RNA can be extracted.
Q: Are preparations for Total RNA form the heart or atrium done by taking the whole tissue or using a piece of it?
A: We use a piece of tissue not the whole tissue.
Q: Does you extract Total RNA plant Arabidopsis from a specific region or the whole Arabidopsis?
A: Total RNA is extracted from the whole Arabidopsis.
Q: Do you send lot-specific COA's that state whether or not a particular RNA lot has been DNase I treated?
A: The customer must request a lot-specific COA that states whether or not the RNA is DNase I treated, otherwise we will only send the general COA.
Q: Do you sell Myeloma tissue RNA?
A: No, unfortunately we are not able to provide Myeloma tissue RNA at this time.
Q: What is the concentration of your Total RNA samples?
A: The Concentration of Total RNAs varies in a range of 1-3 ug/ul.
Q: Can I have the same total RNA from different individuals?
A: Yes. Usually we should be able to provide total RNAs from major organs from at least 10 different donors.
Q: What method did you use for total RNA isolation?
A: The method is proprietary modified phenol extraction.
Q: Is your RNA treated by DNase I?
A: Yes, most of RNAs, especially from the major organs are DNase I treated. However, we don't treat RNAs from rare tissues and some of tumor tissues.
Q: I used your total RNA to do RT-PCR, but I did not get the PCR product?
A: It is possible that the gene of your interest is a very low abundant gene, increase Total RNA amount in your RT reaction or using our mRNA instead may help.
Q: I got your total RNA, and I measured the DEPC water diluted total RNA concentration again by myself, but the concentration I got is lower than yours, can you tell me why?
A: I would suggest you use our method to measure the concentration:
Dilute the RNA in 10 mM Tris-Cl, pH 7.5 but not DEPC water. We usually took 2 ul RNA into 498 ul 10 mM Tris-Cl, (pH 7.5), then measure the OD, use the OD value x 40ug/ml, and then x250 for the final concentration. Using DEPC water will always result lower UV260/280 ratio and lower concentration, and the concentration measured by DEPC water is not stable.
Q: What apparatus was used to homogenize the protein lysate?
A: We used Ultra turrox T25 homogenizer, but any polytron homogenizers or manual homogenizers can be used for this extraction.
Q: Is the phosphorylation state of proteins conserved?
A: Yes, the phosphorylation state of proteins is conserved because we add phosphortase inhibitors.
Q: Are nuclear proteins isolated from total proteins the same way microRNA can be isolated from total RNA?
Q: Does your total protein contain nucleoprotein? If so or not, could you please show us data that present the rate of content?
A: The total protein contains a very minimal amount of nuclear protein, so we don't know the exact portion of the nuclear protein in the total protein, but we know it is very small. If the customer is specifically interested in nuclear protein, we recommend for the customer to buy nuclear protein instead.
Q: Do your protein lysate already contain SDS and have they already been boiled? could you please confirm how these lysates should be treated prior to loading?
A: The protein lysate doesn't have SDS and is not boiled. You need to mix the protein lysate with the loading dye which contains SDS, then boil the lysate for 5 minutes, and put on ice before loading.
Q: Do you have all your total proteins available as non-reduced?
A: All of total protein lysates are native and non-reduced.
Q: Is your total protein extraction kit suitable for extracting from fungi?
A: Yes, the kit can be used for extracting proteins from fungi. The procedure is the same as for extracting tissue proteins.
Q: About total proteins, are you doing a micro-dissection before the extraction or some normal cells around the tumor could be extract with the tumor?
A: We don't do any micro-dissection, and it is very likely some normal cells around are included during the preparation.
Q: Which anti-protease is used in mouse normal tissue total protein extraction?
A: The protein is extracted by our total protein isolation kit. Unfortunately we don't release our protease inhibitor cocktail information.
Q: Is the protein extract from melanoma derived entirely from a primary melanoma, or are metastases included as well? How much normal surrounding tissue is included in the sample for total protein extraction?
A: The melanoma is primary melanoma. The tumor cell percentage of the tissue is about 80%. So there should be about 20% adjacent normal tissue.
Q: How does BioChain measure the amount of protein in your protein lysate?
A: We determine the protein amount by using Bio-rad's Kc kit. Sometimes the protein intensity on the gel is not correspondent of the protein amount very accurately, this is because of the following reasons:
1). The protein need to be resuspended very well every time before loading on the gel, because the protein may not be distributed evenly in the solution. 2). Freezing and thawing may result in protein degradation. 3). Some lysate may have large amounts of small molecule proteins, and the small molecule protein may run out from the gel, that will result in showing less protein on the gel. The best method to follow is to normalize the protein with GAPDH antibody.
Q: Do you have a Total Protein kit for use in Cartilage?
A: BioChain's Total Protein Extraction Kit can be used to extract total proteins from any tissue and cells, including cartilage.
Q: Is this protein lysate prepared with a RIPA buffer?
A: We don't use RIPA buffer, which I understand is sold by Pierce. What we use is Buffer M which includes two ingredients found in RIPA buffer and they are NP-40 and Sodium deoxycholate. Our Buffer M doesn't include Tris-HCl, sodium chloride, and SDS. In addition to NP-40 and Sodium deoxycholate, our buffer includes HEPES (pH 7.9), KCl, EDTA, Sucrose, Glycerol, Sodium Ortho Vanadate, and MgCl2
Q: For your Total Protein Western Blots, how many times can I strip and reprobe any of these blots?
A: It really depends on the protein abundance, for a house keeping gene, such as GAPDH, the blot can be stripped and reprobe for at least 2-3 times
Q: Can you extract protein using PBS only?
A: Yes, we can. But this is a custom made product, when you let us know how much protein lysate you need, we can generate a quotation for you
Q: Can your whole tissue homogenates be used for screening protease activity in specific human tissue samples, i.e. analyzing degradation of particular proteins in these tissues?
A: Our whole tissue homogenates can not be used for screening protease activity in specific human tissue samples since the lysis buffer contains the protease inhibitors which inhibit most of the protease activity. The homogenates can be used for some other enzyme assays, but not for proteases.
Q: What are the components of the total protein lysate buffer? And what is the concentration?
A: The protein lysate buffer includes Hepes, MgCl2, KCl, EDTA, Glycerol, sodium deoxycholate, sucrose, NP-40, and cocktail of protease inhibitors. However the concentrations of the components are proprietary.
Q: Is your total protein ready-to-use? Can I load the protein directly on SDS-PAGE gel?
A: No. The protein need to be mixed with protein loading dye and heat at 95ºC for 5 minutes, then transferred on ice before loading on the gel.
Q: How much protein is used for each spot on the total protein array?
A: Approximately 30 ng.
Q: What is your total protein array like? Does each spot on the array contain each single protein?
A: The total protein arrays are modified nitrocellulose membranes spotted with proteins from different tissues. Each spot on the array contains a protein pool from an organ, but not a single protein.
Q: What method did you use for total protein and compartmental proteins isolation?
A: We used Total protein extraction kit (Cat# K3011010) for total protein isolation, and CNM (K3012010) or CNMCS kit (K3013010) for compartmental protein isolation. Compartmental protein isolation is a patent technology.
Q: How did you measure your protein concentration?
A: We Use Bio-Rad DC protein determination kit to measure the concentration of our protein.
Q: What QC method did you use to prove that your compartmental proteins were well separated?
A: A western analysis with GAPDH (a cytoplasmic protein) antibody shows very strong signal in cytoplasmic protein, but very low signal in nuclear and membrane protein. Histon is shown in nuclear, Na-K ATPase is only shown in the membrane protein, and vimentin is only shown in cytoskeleton protein, very small amount in membrane protein, but not in cytoplasmic and nuclear proteins.
Q: I need to use trypsin to treat the protein before doing my experiment, since you have protease inhibitor cocktail, I wonder if those inhibitors will inhibit the activity of trypsin?
A: Yes, you need to get rid of the protease inhibitors before doing your experiment in such case.
Q: Can I have one kind of protein from different donors?
A: Yes, we can custom make one kind of protein from multiple donors.
Q: My cell's size is small, what needle size should I use in order to break the cells?
A: try needle with gauge size at 32 or 33 because protozoon size is smaller than human cell.
Q: For the CNMCS Compartmental Extraction Kit, what is the best way to store the cells before starting fractionation if the cell culture will not be ready all at once?
A: We recommend for the customer to collect all the cell pellets and keep them in the freezer at -80c, then start to extract until all the pellets are ready.
Q: What is the performance of BioChain's CNM isolation kit with regard to separation of membrane fraction (myofilaments) and nuclei in cardiac tissue? Has this kit been tested on mouse heart tissue?
A: We have not specifically tested the kit's performance for getting rid of myofilaments. We do use our kit to isolate mouse heart nuclear protein, membrane protein as standard protein lysate product, and customers have not had any problems for that. Since we don't have any experience for the myofilaments issue, we can not guarantee it will work. The mechanism for the kit is using hepatonic buffer to release the cytosol protein first, then using high salt buffer to release the nuclear protein, then using a buffer with detergent to release membrane protein.
Q: Can your CNMCS Compartmental Protein Extraction Kit be used to isolate protein from microsome?
Q: I need proteins to make immunoprecipitation. If I use either the CNM or CNMCS kit to extract the proteins, will the protein structure still be intact or will they be denatured?
A: The protein isolated by this kit is native and not denatured, except the cytoskeletal protein.
Q: Could you tell me if this kit has been tested for subsequent use of the sample for immunoprecipitation experiments?
A: Yes, the protein isolated from the kit had been tested for immunoprecipitation, and it worked.
Q: Can the CNM kit be used to isolate membrane fraction by treating envoloped viruses with buffer C, spinning down the viral particles only, and then get membrane fraction from supernatant?
A: The CNM kit can only be applied for tissue and cells; it is very unlikely that it can be applied for virus use. We have never tried this kit for virus protein extraction. We are not sure if buffer C is powerful enough to break the virus, but if it can, then theoretically, you can skip the N step and go to M buffer.
Q: What are the components of the membrane extraction? Do they include plasma membrane and all intercellular membrane such as ER, mitochondira, etc.? Or are they just plasma membrane?
A: The components of the membrane extraction contain plasma membrane and some mitochondria, but no ER or very little.
Q: Will the CNMCS Compartmental Protein Extraction Kit work on plant samples?
A: The kit can not be used for plant samples because the buffer is not strong enough to break the wall of plant cells
Q: What is the concentration of MgCl2 and KCl in buffer C (component K3013010-1) of your CNMCS Compartmental Protein Extraction Kit?
A: The concentration of MgCl2 in buffer C is within 1-10 mM, while 5-40 mM for KCl.
Q: What is the difference between CNM and CNMCS kit?
A: These two kits are exactly the same except CNMCS kit has one more buffer, which is CS buffer for isolation of cytoskeleton proteins.
Q: I am studying mitochondrial proteins, and I would like to know by using your CNM kit, which fraction contains mitochondrial proteins.
A: It is membrane fraction.
Q: What is the components of each buffer in your CNM and CNMCS kit?
A: The information can be obtained from the kit manual.
Q: I would like to know whether the different extractions resulting from CNM kits are adequate for analysis by immuno precipitation? Are the complexes of macro molecules kept for this analysis?
A: Yes, all the proteins extracted by the kit should be ok for i.p analysis. The complexes of macro molecules should be kept.
Q: Do you know if the membrane fraction contains every membrane proteins when you use your CNMCS or CNM kits?
A: Yes it does.
Q: Are all proteins obtained by this CNMCS kit in native conditions or in denatured forms?
A: Cytoplasmic, nuclear, and membrane proteins are in native conditions, but cytoskeleton protein is in denatured form.
Q: Do you have any idea in which fraction the microsome proteins is when you use your CNM or CNMCS kits?
A: We believe the microsome proteins should be in the cytoplasmic protein fraction since the centrifugation speed is not high enough to precipitate the microsome during that preparation.
Q: Can your CNM kit separate the membrane proteins from different organells?
A: It is very hard for us to separate the membrane proteins from different organells, at current situation we don't have any answers for that.
Q: I used your CNM kit isolating compartmental protein, and the protein concentration was measured by UV spectrum. The quantity of the membrane protein was 10 times more than the cytoplasmic protein, is this normal?
A: No, it is not normal. Usually the relative yield of Cytoplasmic and membrane fractions, it's about 2-3:1. I would suggest you run your protein on SDS-PAGE gel and see if the protein intensity matched the concentration you have measured from UV spectrum. If the protein intensity from SDS-PAGE gel is consistent with the quantity measured from UV spectrum, then the tissues or the cells probably were not homogenized very well in the first step. Increasing the speed setting of the homogenizer for tissues, and increasing the repetition of homogenization up to 5 times (normally 3 times). will help. For cells, I suggest pipette enough times to make sure the cells are completely lysed.
Q: Are the proteins isolated by CNM kit good for 2D?
A: Yes, but nuclear fractions need to be dialyzed before using it for 2D experiment.
Q: Can the Dr. P kit be used to extract from fresh blood? How?
A: Yes. You should follow the steps in the user manual, except there is no need to homogenize the blood sample because the solution can lysis the sample directly.
Q: When measuring the concentration of nucleic acid (DNA or RNA), I obtained various concentrations from the spectrometer. Is this normal?
A: The concentration can vary within a certain range. Please dissolve the RNA or DNA completely in 10 mM Tris buffer, pH 7.5. This will help.
Q: Is the protein that I get from your Dr. P Set total protein?
A: Yes, it's total protein.
Q: Can I extract DNA/RNA/Protein from mammalian cells using the Dr. P Kit?
A: Yes, it can be used to extract all 3 components from mammalian cell.
Q: Can your Dr. P Kit be used to extract RNA from leukocyte and bone marrow?
Q: Can your Dr. P Kit be used to extract RNA from leukocyte and bone marrow?
Q: What is Dr. P set product?
A: Dr. P set includes 50 ug RNA, 10 ug genomic DNA, and 100 ug total protein. All these 3 items are isolated from the same piece of biomaterial simultaneously.
Q: What is the difference between Dr. P genomic DNA and general genomic DNA?
A: General genomic DNA was isolated from tissues or cells only while no RNA and protein were isolated at the same time. The quality of general genomic DNA is the best. Dr. P genomic DNA was isolated while RNA and protein were also isolated. There is some difference in the isolation methods.
Q: How much protein is loaded on the gels?
A: Our Western Blot contains 50 µg of tissue lysate per lane.
Q: What type of gel is used? How are proteins transferred?
A: The tissue lysates are run on 4-20% denaturing polyacrylamide gels and electrophoresed. After electrophoresis, the gel is transferred on to a PVDF membrane. This blot is ready to be probed with polyclonal, monoclonal, or peptide antibodies. The Protein marker is indicated at the left margin of the blot. The upper left corner of the membrane has been cut to provide orientation.
Q: Are the proteins of each lane on the blot from the same donor or pooled donors?
A: Generally, protein of each lane on the blot is from a single donor. However, the protein from rare tissues might be pooled.
Q: I saw your manual of Attoglow Western Enhancing Kit that the enhancer works on PVDF membrane, but does the enhancer work for nitrocellulous membrane?
Q: My membrane was dried after the protein transfer. Will the enhancer still work on my dried membrane?
A: Yes, it does. However, we would recommend you extend the enhancer incubation time from 2 minutes to 5-10 minutes.
Q: What is the minimum amount of sample needed to be able to isolate DNA from mitochondria using your mitochondria isolation kit?
A: Our protocol recommends customers use 100-200mg of tissue. However, downscaling the tissue samples to 10 mg should also work too.
Q: Can the mitochondria isolation kit be used on plants?
A: The mitochondria isolation kit is not suitable for plants.
Q: If starting from total RNA, what's the recommended amount of total RNA to start with to extract microRNA using MicroRNA Isolation Kit?
A: Start with 50 ug of total RNA.
Q: Can the Genomic DNA extraction kit be used to isolate DNA from paraffin embedded tissues?
A: Yes, but the tissue section first needs to be deparafinized. Also, the quality of the DNA is very poor compared to using fresh tissue (100-1000 times poorer than from fresh tissue).
Q: Can the Genomic DNA extraction kit be used on plants?
A: Yes, the Genomic DNA extraction kit can be used on plants.
Q: Has your EPO ELISA Kit been validated on human plasma samples?
A: Yes, it has.
Q: What products do you provide that relate to diagnosis research?
A: ELISA kits, QPCR and qRT-PCR products, and BioAssay kits.
Q: Does the ELISA kit come with strip wells?
A: No, the plate does not come with strip wells.
Q: What homology does your Human beta Actin cDNA probe have with rat?
A: The human beta actin cDNA probe we have share 89% homology with rat.
Q: What is the advantage of RapidSeq RNA Sample Prep Kits?
A: Compare with other RNA sample prep kits on the market, the major advantages of RapidSeq Small RNA Sample Prep Kit is the minimum amount of total RNA required for library construction; for RapidSeq mRNA Sample Prep Kit, it is the directional construction feature that distinguishes it from other competitors.
Q: What is the minimum amount of total RNA required for your RapidSeq Samll RNA Sample Prep Kit?
A: The minimum total RNA amount this kit required is 100 pg.
Q: How much purified mRNA should I start with the RapidSeq mRNA sample prep kit?
A: You need to have 50-100 ng purified mRNA to start with the sample prep.
Q: How to fragment the mRNA using the RapidSeq mRNA sample prep kit?
A: You may fragment mRNA buy mixing 2 ul 10X T4 PNK buffer with 16 ul mRNA, heat at 94°C for 5 min, then place the tube on ice. After then add 2 ul 100 mM EDTA to the reaction mixture.
Q: How long it takes using your RapidSeq Samll RNA Sample Prep Kit to construct the sequencing library?
A: It takes less than 7 hours, and total hands-on time is less than 2 hours. This is one of the advantages of the RapidSeq kit.
Q: I have degraded mRNA samples to sequence, how do I optimize the sequence result using your sample prep kit?
A: Under condition of shorter than normal mRNA sizes, decreasing fragmentation time definitely helps to maintain mRNA fragments from further breaking down, which in turn increases chances for better sequencing quality. You may try to use 45 min or 30 min for fragmentation step instead of 60 min, depend on the degree of your mRNA degradation. If you are worrying about the degradation of the RNA from certain source (for example, FFPE tissue), you may skip the fragmentation step and start from the ligation step.
Q: I have a total RNA sample with very low concentration, is there any way to help to enrich it using your sample prep kit?
A: Our test data shows that increasing 1-3 cycles of PCR on the amplification step will help to enrich sample species while keeping the bias level under control. Although more PCR cycle noticeably increases the data bias in DNA sequencing, for RNA sequencing there is more tolerance for this type of tweak.
Q: What volumes should we pool together and how much do we load on the gel and how much gel loading buffer should we use?
A: If your are making 2 samples with different aligners, then you will need to pool 25ul of each reaction for a total of 50ul. If you are making 4 samples with 4 different aligners, then you will need to pool 12.5ul of each sample for a total of 50ul. The goal is maintain a final volume of 50ul because you will need to add 10ul of loading buffer. Finally, you will load 25ul of the pooled sample with loading buffer to one well and another 25ul to a different well. You will have 10ul left over, but that is okay because you should have plenty to sequence.
Q: Can you provide the adapter sequences for the data analysis purpose?
A: Sure, we are going to provide the specific adapter sequences upon request from our customers.
Q: What type of sequencing service BioChain offers?
A: BioChain provides cost-effective, high quality DNA and RNA sequencing service which is suitable for all your one-stop shopping or customized project needs. Based on our Illumina platforms, we provide a wide spectrum of service such as de novo sequencing, whole genome resequencing, targeted sequencing, and whole transcriptome profiling. We will work with you from the beginning of your project and until you receive the analyzed sequencing data from us.
Q: What is UltraScript RT?
A: UltraScript RT is a thermostable, genetically engineered reverse transcriptase which BioChain purified to near homogeneity by FPLC to ensure high thermal stability, high specificity, high fidelity, high yield and more full length cDNA synthesis that the premium reverse transcriptase provides.
Q: What is the reaction temperature for UltraScript RT?
A: The optimal fist-strand cDNA synthesis temperature for this enzyme is 60 °C, and it has a broad working temperature range from 37°C to 65°C.
Q: How big the size of cDNA can the UltraScript RT synthesize?
A: Up to 12 Kb.
Q: What is the largest size of cDNA that your M-MLV RT synthesizes?
A: Up to 5Kb.
Q: What is minimum amount of RNA can the UltraScript RT synthesize into cDNA?
A: As low as 1 ng.
Q: Can I use the UltraScript RT to replace SuperScript III RT in my assay?
A: Yes, definitely. We have tested the performance for both enzymes side by side, and both enzymes worked the same way under various conditions.
Q: Can your optimax cDNA synthesis kit be used as direct tissue to cDNA synthesis, or direct cell to cDNA synthesis?
A: No. It has to be started from total RNA or mRNA.
Q: What type of hotstart taq enzyme do you provide?
A: With licenced technology from Applied Biosciences, BioChain provides chemically modified form of Taq DNA polymerase which is in an inactive state, and by heat activation, the chemical modifier is permanently released, regenerating highly active enzyme.
Q: What is the advantage of chemically modified hotstart enzyme?
A: The chemically modified Taq enzyme can be activated partially or completely in a pre-PCR heat step, or can be activated slowly in a time-release manner during the denaturation steps of thermal cycling. With or without a limited up-front heat activation step, active enzyme is released slowly during thermal cycling to match template concentration and increase specificity. The chemical hotstart enzyme is also free of biological contamination due to the chemical modification method.
Q: What is EvaGreen and why do you have it in the qPCR mix instead of SYBR Green?
A: EvaGreen dye is a next-generation DNA-binding dye with features ideal for use in real-time PCR. Compare with SYBR Green, it has better dye properties such as less PCR inhibition, better stability and wider fluorescence spectra. The net results of application of EvaGreen dye in qPCR SuperMix are higher resolution melt curve (HRM) and lower Ct value at the same PCR condition.
Q: What type of hotstart taq enzyme do you provide?
A: With licenced technology from Applied Biosciences, BioChain provides chemically modified form of Taq DNA polymerase which is in an inactive state, and by heat activation, the chemical modifier is permanently released, regenerating highly active enzyme.
Q: I did run qPCR with the Master Mix. However, no amplification was seen, why?
A: Please check your program setting and see if the time in first step of 95°C activation is 10 minutes. Chemically modified hotstart enzyme requires minimum activation time of 10 minutes, any time shorter than that will lead to problem of amplification.
Q: How long of a fragment your qPCR superMix can amplify?
A: The standard amplicon size of BioChain's qPCR SuperMix is 800 bp.
Q: Is your Taq enzyme or SuperMix compatible with any type of real-time PCR instrument?
A: Yes, BioChain's real-time PCR reagents are compatible for most of the qPCR instruments with standard block. However, if you are using Bio-Rad's iCycler iQ, iQ5, MyiQ, and MyiQ2 systems, you need to add Bio-Rad's Fluorescein Calibration Dye (cat#170-8780) for calibration purpose.
Q: How many matched pairs do you have for each tumor type?
A: We usually have more than 10 different donors.
Q: Do you provide custom made FISH probes?
A: Unfortunately, we do not provide this service at this time.
Q: Are your samples from surgery or death donors? Do you provide information about sex, age and about the surgery/post-mortem time elapsed before processing the tissue?
A: The normal tissues are from death donors. We do provide sex and age information. The post-mortem is about 4-6 hours. The tumor tissues are from surgery, and they are snap frozen with liquid nitrogen right after dissected from the body.
Q: What's the species of your bovine products?
A: We don't know the exact bovine species (also called breed). We did check with the vendor regarding this issue for other customers, but the vendor was not able to give a clear answer. The bovine product could be from any breed that is raised in the United States. A few of these breeds are Hereford, Brahma, Charolais, Black Angus, Simmental, Limousin, and Holstein mix. We have no way to know the exact breed of these bovine products.
Q: What is species of your monkey products?
A: We have Rhesus (Macaca Mulatta) and Cynomolgus monkey. There is suffix "cy" in the end of catalog numbers of Cynomolgus monkey products.
Q: What is the species of your mouse products?
Q: What is the species of your rat products?
A: Sprague Dawley, Wistar
Q: What is the species of your dog products?
Q: Which part of tissue do you isolate RNA from plants?
- Q: Can the microRNA extraction kit be used to isolate microRNA from mouse plasma? If so, what is the amount of plasma required and what is the average yield?
- A: We have not tried this with the microRNA extraction kit. We believe the amount would be minimum and doubt you would get anything from the plasma.
- Q: Is the microRNA isolation kit suitable for use in cell culture supernatant?
- A: Our kits are suitable in blood and serum. Since blood and serum refers to cell culture supernatant the answer is yes.
- Q: What is the yield using BioChain's microRNA isolation kit?
- A: The yield is low and depends on tissue type. For high yield tissue such as liver, the estimated microRNA yield is around 1-5 ug per gram.
- Q: What's the concentration of Chloroform and Acidified Phenol? Regarding Acidified Phenol:Chloroform, is Phenol: Chloroform: Iso-amyl alcohol(25:24:1)?
- A: 70% phenol and 30% chloroform
- Q: The customer would like to use this kit for blood samples. Please inform us of the citation or any information if known.
- A: A citation is not available yet because the kit was just upgraded for blood sample microRNA isolation.
- Q: How is the disproportionate rate of yield of miRNA from the same sample (e.g. same tissueof the same individual)? If you have any data, please let us know.
- A: microRNA yield is very low and usually is not measurable or quantitative by UV, from 1ml blood, the microRNA yield is about 1-15 ng.
- Q: The customer would like to know the recommended internal standard gene for real-time PCR of blood sample miRNA.
- A: mi24 and mi16 should be OK.
- Q: "BioChain’s microRNA qRT-PCR primer sets were uniquely designed and optimized for detecting mature miRNA with high specificity and sensitivity at low PCR annealing temperature. I am confused by "low PCR annealing temperature" in this description. Is this a advantage or key point to detect only mature miRNA, separately from precursor miRNA? Please let me know why low annealing temparature is good.
- A: Usually if the annealing temperature is low, then the specificity will drop. But biochain can still keep the high specificity with low temperature.
- Q: I would like to measure COX activity of mouse soleus muscle with this kit. Can you recommend a sample preparation (e.g. homogenate)?
- A: Try the Mitochondria Isolation Kit.
- Q: Lot A810233 - I used the included control but didn´t get a change in absorption. OD was the same after 5, 10, 20 and 30 seconds. I added the corresponding reagents and started directly the reading.
- A: The customer should mix the sample within 5 sec and start the reading right away. Starting the reading at the time point of 20sec is too late. The cytochrome C oxidase reactions is a second-order reaction and usually occur very quickly in the first 15-20sec, then slow down.
- Q: Can the Mitochondrial Activity Assay Kit be used on sheep oocytes? How small a sample it can work with, in terms of amount of tissue? My customer would like to analyze individual oocytes separately - is the kit *that* sensitive?
- A: We have not tested the mitochondria activity assay kit on sheep occytes. However, we assume it's applicable since it states in the user's manual, "Mitochondria can be prepared from a variety of mammalian cultured cells and tissues by differential centrifugation." The user's manual doesn't indicate exactly how much starting material the customer should use but it does state "Assay various amount of samples to find a linear range for the sample." For optimal preparation of mitochondria, it is recommended to use the BioChain’s Mitochondria Isolation Kit (Catalog # KC010100).
- Q: Is the mitochondria activity assay kit suitable for use by microplate?
- A: We have no protocol to show that our kit can be applied to microplate. Please ask the customer to modify it own their own.
- Q: Is BSA (Bovine serum albumin) included in any component of the mitochondria isolation kit?
- A: No.
- Q: How much concentration of sodium is included in the Lysis Buffer?
- A: The sodium concentration is very low in the buffer (less than 1 mM).
- Q: Can the mitochondria isolation kit be used on plant?
- A: No, the mitochondria isolation kit is not suitable for a plant.
- Q: What is the smallest amount of sample she'll need to be able to isolate DNA from mitochondria using our mitochondria isolation kit?
- A: Our protocol recommends customers to use 100-200mg tissues. However downscaling the tissue samples to 10mg should work too.
- Q: Which reagent does she need to extract DNA from mitochondria? Does this reagent come with the mitochondria kit?
- A: The mitochondria kit doesn't come with DNA extraction reagents. The customer will need to work out the mitochondria DNA extraction protocol. Refer to the website for mitochondria DNA isolation http://wheat.pw.usda.gov/~lazo/methods/lazo/dnaplmit.html
- Q: For how long can the mitochondria be stored after extraction?
- A: The stability of the mitochondria isolated from different tissues varies. I can tell the mitochondria isolated from human kidney can still keep 80% of the activity after 6 months frozen in -75C
- Q: Is BioChain's mitochondria isolation kit suitable for use in White Blood Cells (WBCs)?
- A: Yes.
- Q: Is the Mitochondria Isolation kit suitable for use with cryo preserved tissue?
- A: Yes.
- Q: Can peroxisomes be isolated from tissue or cells?
- A: We do not have the data.
- Q: Is it possible to obtain the citosolic fraction of the cells after any of the centrifugations in the protocol?
- A: The kit can be modified for cytoplasmic isolation.
- Q: I would like also to know if it is possible to use the RNA of the extracted mitocondrias to perform RT-PCR?
- A: If you can isolate RNA from the extracted mitochondria, then you can use the RNA for RT-PCR with random hexmer for RT (oligo dT might not work because mitochondria RNA may not have poly A region in the 3' tail)
- Q: Is the mitochondria isolation kit suitable for isolating mitochondrial from human primary blood monocytes?
- A: Though we did not have experience using this kit for blood monocytes, theoretically the kit should work.
- Q: After collection of blood sample, how many days we can store the blood for mitochondrial isolation and activity assay. Please provide me the storage conditions and time of the sample.
- A: If stored in -80C, at least one year.
- Q: For the customer it is crucial that the mitochondria are still intact after isolation, i.e. the citrate cycle should still be functional. The customer says that e.g. cytochrome c still being detectable inside isn’t really an indicator for intact mitos because it is located at the inner membrane, meaning the outer membrane could be destroyed anyway. Have you performed other tests showing that the mitos isolated with your kit are still intact and could you share the data?
- A: Ask the customer to test membrane integrity which is also included in the kit.
- Q: Is the mitochondria isolation kit suitable for isolation of mitochondria from human hair sample and mouse tissue.
- A: Technically it should be but this experiment has not been performed by us.
- Q: Can other membrane proteins be isolated with this kit? In the user's manual it reads, “Mitochondria can be frozen at -80°C until use.” How long can they be stored without being damaged/losing performance? When you store them at -80°C, do they keep their structure AND their function?
- A: No, other membrane proteins cannot be isolated with this kit. It's best to use CNM for other membrane proteins. The longest we tested was after a month, the activity lost about 10%.
- Q: Using both Mitochondria Isolation Kit & Genomic DNA Extraction Kit, is it possible to isolate mitochondrial dna?
- A: I confirm that by using both BioChain’s Mitochondria isolation kit and genomic dna extraction kit, it's possible to isolate mitochondrial dna. Though I should note that the yield will not be very high as I believe only 1% of mitochondrial dna makes up the total genomic dna. To give an example, let say the customer has 1 gram of tissue and from this tissue is able to isolate about 100 ug of genomic dna. Then, the expected yield is about 5 ug of mitochondrial dna.
- Q: I have a customer interested in the Mitochondria Activity Assay Kit but he wants to know if he also needs to get the Mitochondria Isolation kit or if he can simply lyse the cells and run the Activity assay? This information was not clear by reading the package insert.
- A: If the method of lysating the cell is for total protein extration, then the customer doesn't need to purchase the mitochondria isolation kit. However, if for the purpose of mitochondria isolation, then it will not work to only purchase the mitochondria activity assay kit. For this purpose, it's necessary to purchase the mitochondria isolation kit as well.
- Q: With reference to Cat No: KC01100 (Mitochondrial DNA Extraction Kit), Cat No: K5082100 (DNA Methylation Detection Kit) although the kit mentions that DNA can be extracted form various tissues and cultured cells, whether we could extract mitochondrial DNA from sperm cells and also DNA methylation could be performed in sperm cells?
- A: First, you have to carefully read the kit name, it is mitochondria isolation but not mitochondrial DNA isolation. You can isolate mitochondria from sperm cells but not mitochondrial DNA from sperm cell. But I think when you get the mitochondria, you can use other kit for the DNA isolation. You can always use our DNA methylation detection kit to detect any DNA methylation.
- Q: I use fresh hepatic tissue for mitchondria isolation to measure cytochrome C oxidase activity. If I would like to measure the mitochondria protein concentration, what steps I should take. On your User’s Manual and Instructions’ protocol, you mentioned “Resuspend the pellet in 50 – 100 μl mitochondria storage buffer and keep it on ice before downstream processing.” On the other hand, “If mitochondria protein lysate is desired, resuspend the pellet in 100 μl Lysis Buffer with 1x protease inhibitors. The mitochondria protein concentration can be measured at this step. The expected concentration should be about 0.5 – 1 mg/ml. Store the lysate at -80°C.” I am a bit confused, if I measure the mitochondria protein concentration, can I skip the step “Resuspend the pellet in 50 – 100 μl mitochondria storage buffer and keep it on ice before downstream processing.” Or I should spin down the “Resuspend the pellet in 50 – 100 μl mitochondria storage buffer”, then resuspend the pellet in 100 μl Lysis Buffer with 1x protease inhibitors. Please let me know what steps I should take when I would like to measure the mitochondria protein concentration and the cytochrome C oxidase activity. And can I measure the mitochondria protein concentration after store the resuspend the pellet in 100 μl Lysis Buffer with 1x protease inhibitors at -80°C?
- A: If you need to measure Cytochrom C activity, then you need to resuspend the pellet in mitochondrial storage buffer. If you need to have mitochrondrial protein lysate, then you need to resuspend the pellet in the lysis buffer with protease inhibitor. If your experiment is just for mitochondrial protein, such as using the mitochondrial protein lysate to do Western Blot, then you can skip the storage buffer and resuspend the pellet in the lysis buffer directly. If you want to do both, then I recommend you split the solution after step 3, then after spin down in step 4, you should get two pellets, you resupend one pellet into storage buffer, and resuspend another pellet into lysis buffer.
- Q: 1) Concerning Isolation Kit, he would like to know whether this kit can be used for myxomycetes sample. 2) If your kit can isolate mitochondria of myxomycetes sample, could these mitochondrial activity can be measured by KC310100?
- A: The KC010100 kit (Mitochondria Isolation Kit for Tissue and Cultured Cells) will work for myxomycetes sample only if the first step of homogenization changed from polytron tissue disruptor/dounce tissue grinder to glass beads vortex. The reason is fungi need much more shearing force to break the cells up.
- Q: Regarding the whole cytosolic component, not the mitochondrial cytosol, how do I process the cytosolic bit which is the supernatant to get the proteins? Do I have to spin again the supernatant? and if i do, and what speed?
- A: The supernatant from the 12,000 g centrifuge can be treated as cytosol, and you don't need to spin it again. You can measure the protein concentration from the supernatant and see how much protein there.