Frozen Tissuex

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.

Paraffin Tissuex

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?
A: Yes.

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?
A: Formalin.

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.

Genomic DNAx

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.

cDNAx

Q: Are all your cDNAs from total RNAs that have gone through DNase I digestion?
A: Yes.

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 betested 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.

RNAx

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.

Protein lysatex

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 phosphatase inhibitors.

Q: Are nuclear proteins isolated from total proteins the same way microRNA can be isolated from total RNA?
A: No.

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 DC

DNA Extraction Kitx

Q: Can the Genomic DNA extraction kit be used on plants?
A: Yes, the Genomic DNA extraction kit can be used on plants.

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 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 leukocyte 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.

Compartmental Protein Isolation Kitx

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 dont 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?
A: No.

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.

Dr. P Kitx

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?
A: Yes.

Q: Can your Dr. P Kit be used to extract RNA from leukocyte and bone marrow?
A: Yes.

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.

Western Blotx

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?
A: Yes.

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.

NGS Reagents and Servicex

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

Reverse Transcriptase and cDNA Synthesis Kitx

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

PCR and qPCR Mixx

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

Micro RNA Related Kitsx

 

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.

Mitochondria Related Kitsx

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: I used the included control but didn

Othersx

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.