Transfection experiment skills (the following information is all provided by the customer) DNA transfection reagent How to prepare the plasmid DNA required for transfection Transfection efficiency is affected by many factors. In addition to factors such as cells, culture media and carriers, another very important factor is the quality of DNA. In order to compare the transfection efficiency of different manufacturers, we purchased plasmid preparation kits from three different suppliers to prepare pEGFP-N3 plasmid DNA, and transferred the prepared pEGFP-N3 plasmid to NIH-3T3 cells by different methods. The pEGFP-N3 plasmid was prepared by Endofree Maxi Piasmid Kit (Qiagen), PerfectPrep Endofree Piasmid Maxi Kit (5 PRIME, inc) and NucleoBond DNA Maxi Kit (MACHEREY-NAGEL GmbH & Co. KG), and we measured absorbance at 230, 260, 280 and 320 nm. The quality of DNA. The DNA quality prepared by the above three methods was as follows: MACHEREY-NAGEL>5Prime>Qiagen. pEGFP-N3 was shipped to NIH-3T3 cells by PolyJet DNA transfection reagent. The transfection efficiency was consistent with the quality of DNA. The transfection efficiency of MACHEREY-NAGEL purified DNA was the highest, while that of Qiagen purified DNA was the lowest. Therefore, the plasmid preparation kit of MACHEREY-NAGEL GmbH & Co. KG is considered to be the best choice for preparing transfection grade DNA. How to prepare transfection grade plasmid DNA Transfection efficiencies are affected by a variety of different parameters. Besides factor such as cell culture, medium and vectors, one of most critical parameters is DNA quality. We prepared pEGF-N3 plasmid DNA by large plasmid preparation kits from three different vendors and tested transfection Efficiencies by delivering pEGFP-N3 DNAs prepared by different methods to NIH-3T3 cells. Then pEGFP DNA was delivered with PolyJetTM (Cat # SL100688) DNA transfection reagent to NIH-3T3 cells per manufacturer's protocol. In accordance with DNA purity results, we found that DNA from MACHEREY-NAGEL gave the best transfection efficiency while Qiagen DNA gave the Worsiness preparation. from MACHEREY-NAGEL GmbH & Co. KG is confirmed to be the best choice to prepare tansfection grade DNA. Transfection of epidermal cells Epidermal cells are widely distributed throughout the body, and normal epidermal cells are more difficult to transfect, especially using liposome-based transfection reagents. We transfected normal human colonic epithelial cells using the Amaxa method and obtained 65% of GFP-labeled cells. Thanks a lot to SignaGen, now we can successfully transfect normal human colonic epithelial cells with GenJet Ver II and the transfection efficiency is significantly improved to 75%. Here are the simple steps to transfect epidermal cells with GenJet Ver II (Cat#SL100489): 1. Ensure that the epidermal cells reach 85% confluence during transfection and ensure that the cells are fresh and healthy. 2. For 6-well plates, dilute 1.0 μg, DNA and 3.0 μl, GenJet Ver. II (Cat=SL100489) with serum-free DMEM. Add the diluted transfection reagent to DNA and leave it at room temperature for 15 minutes. A transfection complex is formed. 3. The transfection complex was directly added to the surface cells: 6-well plates containing 1.0 ml of medium per well, transfected in the presence of serum/antibiotics. 4. After 5 hours of transfection, the transfection complex was cleared and replaced with normal growth medium. 5. The expression of the transfected gene was detected 24-48 hours after transfection. Transfection of epithelial cells. Epithelial cells are found throughout the body, from skin to glandular formations within tissues. In vivo these cells are attached to a three dimensional basement membrane matrix. Normal epithelial cell is extremely hard to transfect especially to liposome based transfection reagents. We ever used electroporation ( Amaxa) to transfect normal human colonic epithelial cells and got 65% GFP+ cells. Thanks to SignaGen, now we have successfully transfected normal human colonic epithelial cell with GenJet Ver. II with up to 75% GFP+ efficiency. The following is a brief protocol for Transfecting epithelial cell with GenJet Ver. II (Cat # SL100489): 1. Grow epithelial cell ~85% confluency at time of transfection. Epithelial cells must freshly prepared and healthy. 2. For 6-well plate, dilute 1.0 μg of DNA and 3.0 μl of GenJet Ver. II (Cat # SL100489) per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes . 3. Trans transfection is present in serum of antibiotics with transfection volume of 1.0 ml per well of 6-well plate. 4. Remove transfection complex 5 hours after transfection and change back to normal growth medium. 5. Check transgene expression 24~48 hours post transfection. Choose more effective experimental steps for specific cells GenJet Ver. II, LipoD293 and PolyJet DNA transfection reagents for mammalian cells provide you with two different transfection steps - general and advanced steps - for different mammalian cells. The advanced steps are primarily directed to difficult mammalian cells such as MDCK, MDA-MB231, Caco-2 and the like. If the transfection efficiency is less than 5% using a typical transfection step, the transfection efficiency can be increased to 60% using the advanced transfection step. How you choose the transfection step depends on your cell characteristics. For commonly used cells such as HEK293, Hela, CHO, 3T3, COS, HepG2, LNCaP, PC3, PC12, U2-OS, L929, MCF-7 and Huh-7, the general experimental procedure can obtain satisfactory transfection results. For difficult cells such as MDCK, MDA-MB231, Caco-2, SaoS-2 and HUVEC, advanced transfection steps can be used. For your cells, if you don't explicitly choose which transfection step, we recommend that you try the general steps first. If you use the general procedure to achieve a transfection efficiency of less than 5%, then your cells are more difficult to rotate. Try the advanced transfection step. Choose a more efficient protocol for your specific cell. GenJet Ver. II, LipoD293 and PolyJet DNA in vitro transfection reagent all offer two protocols for transfecting mammalian cells--------a general protocol and an advanced protocol. The two protocols are written for transfecting different types of mammalian cells. The advanced protocol which involves a shaving cell technology is for very hard to transfect mammalian cells like MDCK, MDA-MB231, Caco-2, etc. The general protocol usually gives less than 5% efficiency while advanced protocol can get up to 60% efficiency On these problematic cells. The principle to decide which one protocol is used to your cell is highly dependent upon the nature of your cell. For commonly used cells like HEK293, Hela, CHO, 3T3, COS, HepG2, LNCaP, PC3, PC12, U2-OS, L929 , MCF-7 and Huh-7, be sure to use general protocol which usually gives very good transgene expression while the advanced protocol does not help. For very hard to transfect cells like MDCK, MDA-MB231, Caco-2, SaoS-2 And HUVEC, follow the advanced protocol by trypsinizing these adherent cells first followed by incubation of the cell pellet with transfection complex. If you do not know for your specific cells how to choose the more efficient protocol, we suggest you try general protocol first. You get less than 5% efficiency with the general protocol, then your cell is hard to transfect in nature and you can consider trying advanced one. How to optimize PolyJet transfection reagent Transfection of epidermal cells and Raw267.4 cells with PolyJet DNA transfection reagent was very effective and minimally toxic. I would like to share the following points on how to better optimize PolyJet transfection reagents: 1. The quality and quantity of DNA. The purity of DNA is critical for transfection experiments. DNA prepared by E Coli, A260/280 must reach 1.80 or higher. For a 6-well plate, typically ~1.0 μg of DNA per well. For other cell culture dishes, the DNA content can be adjusted according to the surface area. 2. The ratio of PolyJet/DNA. For transfection of the epidermis and Raw267.4 cells, we fixed the ratio of PolyJet/DNA at 3:1 and obtained satisfactory results without worrying about finding a more suitable ratio. 3. Dilute. Do not use serum for dilution of DNA and PolyJet. We used a serum-free high-dose DMEM medium that worked well. Please do not choose Opti-MEM because it can affect the formation of transfection complexes. Therefore, do not use Opti-MEM to dilute plasmids and PolyJet. 4. Transfection can contain serum. We performed transfection experiments in the presence of serum/antibiotics and both, and did not find any difference in transfection efficiency between the two cases. Therefore, serum/antibiotics have no effect on PolyJet transfection reagents. Some technical tips for optimizing PolyJet reagent. PolyJet DNA Transfection Reagent is very efficient to transfect epithelial and Raw 267.4 cells in our hands without significant toxicity. Here I would like to share several technical points for better using PolyJet transfection reagent. DNA prepared from E Coli. must be 1.80 at A260/280 or higher. For 6-well plate, ~1.0 μg of DNA per well is usually good enough. For DNA. Other cell culture format, just adjust DNA amount per culture dish's surface area. 2. Ratio of PolyJet/DNA. For epithelial and Raw 267.4 cells, we locked the ratio at 3:1 with excellent results and never bothered to find the optimal ratio. 3. Diluent. Diluent used to dilute DNA and PolyJet reagent must be serum free. Serum-free DMEM medium with high glucose works for us. Opti-MEM is NOT a good choice because it may affect formation of transfection complex. So never use Opti -MEM as dilute. 4. Transfection with serum. We performed transfection with or without serum/antibiotics and failed to find any difference in efficiency. Therefore, PolyJet reagent is NOT interfered by serum/antibiotics. How to optimize LipoD293 transfection reagent LipoD293 DNA Transfection Reagent is an upgraded version of the liposome DNA transport tool. Our laboratory successfully transfected HepG2, LNCaP, CHO and HEK293 cells using LipoD293 DNA transfection reagent. Next, we are happy to share with you tips on how to improve transfection efficiency and reduce toxicity. 1. The ratio of LipoD293/DNA. Although the appropriate ratio is determined by the cell type, using LipoD293/DNA, a fixed ratio of 3:1 usually gives good transfection efficiency. 2. The amount of DNA in each well. For 24-well plates, I use 0.2 to 0.5 μg of DNA per well. Too much DNA (for example, 1.0 μg per well) is not necessary and produces high toxicity. Other cell culture vessels can adjust the DNA content according to the surface area. 3. Dilution of DNA and LipoD293. Never use serum-containing media to dilute both. High-sugar Plain DMEM medium is a good choice, but high sugar is not very important. Never use Opti-MEM (invitrogen)! Opti-MEM can affect the formation of transfection complexes. My colleague did not realize the importance of this point and mistakenly used Opti-MEM, resulting in inefficient transfection. 4. The presence or absence of serum/antibiotics has no effect on transfection. Currently, the mammalian cells we are using are usually transfected with LipoD293, and serum/anti-purification has no effect on transfection efficiency. Unlike other liposome-dependent transfection reagents, LipoD293 is not affected by serum/antibiotics, so you don't have to worry about the high cell death rate caused by serum culture in transfection. 5. Change the medium after transfection. For the mammalian cells we are studying, usually, we change the medium 24 hours after transfection, and it is not necessary to change the medium 5 hours after transfection. Some points which are important but ofetn neglected for optimizing LipoD293 reagent. LipoD293 DNA Transfection Reagent is an enhanced version of liposome DNA delivery tool. Our lab has been using LipoD293 DNA transfection reagent for transfecting HepG2, LNCaP, CHO and HEK293 cells with very good lucks. 1. Ratio of LipoD293 reagent/DNA. While the optimal ratio is cell type dependent, the ratio of reagent/DNA locked at 3:1 often gives excellent efficiency. Our labs has been transfecting HepG2, LNCaP, CHO and HEK293 with LipoD293 reagent at 3:1 ratio with very good efficiency. For 24-cell plate, I used 0.2 to 0.5 μg of DNA per well. Too much DNA (eg, 1.0 μg per well) is unnecessary and might lead to high cytotoxicity. For other cell culture format, Adjust DNA amount per culture dish's surface area. 3. Diluent for diluting DNA and LipoD293 reagent. Diluent must be serum free. Plain DMEM medium with high glucose is usually very good. High glucose seems not that important. Never use Opti-MEM (from Invitrogen)! This is very important 'cause My colleagues often failed to recognize importance of the diluent and misused Opti-MEM, leading to suboptimal efficiency. Per our initial test, Opti-MEM is able to disrupt formation of transfection complex. 4. Transfection with or without serum/antibiotics. For all mammalian cells we are currently working on, we always perform transfection with LipoD293 reagent in the presence of serum (10% FBS)/antibiotics without compromising any efficiency, thus greatly simplifying our transfection procedures Unlike other liposome based reagent, LipoD293 reagent is usually NOT interfered by serum/antibiotics, so do not bother to perform transfection in serum-free medium which otherwise results in high cell death. Medium change post transfection. We usually change medium 24 hours after transfection. Medium change 5 hours post transfection is not necessary for all the mammalian cell we are currency using. Simple steps to transfect L929 cells L929 is a mouse fibroblastoma cell line that is frequently used to detect TNF-alpha and TNF-beta. Treatment of TNF often causes apoptosis and death, so L929 cells are often used as immunoassays. However, transfection of L929 cells is very difficult, especially with liposome-based transfection reagents. We used GenJet VerII and PolyJet to transfect L929 cells to achieve 75% transfection efficiency. The simple experimental procedure is as follows. 1. Ensure that the L929 cells reach 80% confluency during transfection, the cells must be healthy and the passage must not exceed 9 generations. 2. For 6-well plates, dilute 1.0 μg, DNA and 3.0 μl, Genjet VerII or PolyJet transfection reagents in serum-free DMEM. The diluted transfection reagent is added to the DNA and allowed to stand at room temperature for 15 minutes to form a transfection complex. The cell culture vessels of other specifications can appropriately adjust the DNA content according to the surface area. 3. The transfection complex was directly added to L929 cells; 6-well plates containing 1.0 ml of medium per well were transfected in the presence of serum/antibiotics. 4. Monitor the expression of transfected genes 24 to 48 hours after transfection. Brief procedures for transfecting L929 cell. L929 is a murine aneuploid fibrosarcoma cell line which is often used to assay TNF-alpha and TNF-beta. Treatment with TNF initiates apoptosis and subsequent cell death, therefore L929 cell is often used for immunological assays. However L929 cell is hard to transfect, Especially resistant to liposome based transfection method. We have been using GenJet Ver. II and PolyJet to transfect L929 cell and got up to 75% efficiency. The brief procedures are described below for transfecting L929 cells with GenJet and PolyJet. 1. Grow L929 cell to ~80% confluency at the day of transfection. L929 cell must be healthy and less than 9 passages for maximum efficiency. 2. For 6-well plate, dilute 1.0 μg of DNA and 3.0 μl of GenJet Ver. II or PolyJet reagents per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes. Other format of cell culture formats, scale down or up per surface area of ​​culture dish. 3. Trans transfection complex to L929 cell directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate. 4. Check transgene expression 24~48 hours post transfection. Tips for choosing a GenJet TM , LipoD293 TM & PolyJet TM DNA Transfection Reagent Dilution Solution The choice of which dilution to dilute the DNA and transfection reagent is critical to the preparation of an effective transfection complex. In addition to temperature and incubation time, the nature of the diluent is also important for the preparation of highly efficient transfection complexes, as well as DNA transfection efficiency. According to our experimental data, the transfection efficiency obtained with the appropriate dilution is at least 50 times the transfection efficiency using the wrong dilution. More importantly, the experimenter has always ignored the importance of the diluent and has never even realized the importance of the correct dilution for the formation of an effective transfection complex. The following experimental techniques can help you choose the appropriate dilution is GenJet TM, LipoD293 TM & PolyJet TM DNA transfection reagent. 1. In order to prepare a more effective transfection complex, do not contain serum or protein in the diluent. 2. Try to use dilutions close to the cell culture medium. For example, if you use HEM293 cells with DMEM (supplemented with serum and antibiotics), the most suitable dilution is DMEM without serum or antibiotics: if you use RPM1 -1640 (supplemented with serum and antibiotics) to culture Hela cells, then the most suitable dilution is RPM1-1640 without serum, antibiotics. 3. Do not use media containing unknown ingredients. For example, do not use Opti-MEM TM diluted GenJet ™, PolyJet ™ and LipoD293 ™ DNA Transfection Reagent. According to our experimental data, if the transfection reagent and DNA were diluted with Opti-MEM, the transfection efficiency for HEK293, Hela, CHO and NIH 3T3 cells was reduced by at least 50%. Opti-MEM TM serum may contain fewer, resulting in greatly reduced transfection efficiency. 4. Serum-free high-dose DMEM is a good choice and can be compatible with other growth media. If you do not get high transfection efficiency, you may consider replacing the diluent. If you are not sure how to choose the right one. For the first dilution, you are advised to try serum-free high-dose DMEM, and you should get satisfactory transfection efficiency. Tricks in choosing diluent for GenJetTM, LipoD293TM & PolyJetTM DNA transfection reagents. For efficient formation of transfection complex, to choose an appropriate diluent which is used for diluting DNA transfection reagent and DNA is essential. In addition to temperature, incubation time, the nature of diluent is very important for complete formation of transfection complex, thus significant affecting Per good validation data, an appropriate diluent can be to 50 times more efficient than a "wrong" diluent. What's more important is researchers often neglect the importance of diluent and fail to recognize role of diluent in the process of transfection complex Formation. 1. For complete formation of transfection complex, the diluent must be serum-free and protein free. 2. Try to use a diluent which is closest to the composition of cell culture medium. For instance, if you are using DMEM (supplemented with serum and antibiotics) to grow HEK293 cells, the best diluent is serum-free and antibiotics-free DMEM If you are using RPMI-1640 Medium (supplemented with serum and antibiotics) to grow Hela cells, the best diluent is serum/antibiotic-free RPMI-1640 medium. 3. Never use a medium which contains unknown components. For example, never use Opti-MEMTM to dilute GenJetTM, PolyJetTM and LipoD293TM DNA transfection reagents. Per our validation data, diluting our transfection reagents and DNA with Opti-MEMTM At least sacrificed 50% transfection efficiency on HEK293, Hela, CHO and NIH 3T3 cells. Also Opti-MEMTM may contains low level of serum which will greatly compromise transfection efficiency. It is very easy accessible and compatible with other cell growth mediums. If you have trouble with lower transfection efficiency, you may think about changing diluent to serum-free DMEM with high Glucose. If you do not know how to choose a proper diluent, you can try serum-free DMEM with high glucose first which usually gives you very good transfection efficiency. Smart Choice for Reporter Gene Detection - LipoD293 DNA Transfection Reagent Use lipoD293 transfection reagent to transfect mammalian cells (such as Hela, PC3) to detect the luciferase reporter gene. Compared to other transfection reagents, you will get more fluorozyme readings and less cell death. Using unsupplemented RPM11640/DMEM instead of Opti-MEM to dilute lipoD293 and luciferase reporters, you will get a higher luciferase reading (doubling) and save even more money. The following are some of the experimental points in the experiment: 1. Use the same type of medium (but unsupplemented) to dilute lipoD293 reagent and luciferase reporter DNA (for example, if the cells are cultured in RPM11640-based medium, then unsupplemented RPM11640 can be used as the dilution solution); 2. There is no need to change the medium before and after transfection; 3. Add the diluted lipoD293 reagent to the diluted DNA, mix and incubate for 15-20 minutes at room temperature. Smart choice for reporter assay with LipoD293 DNA transfection reagent. Use lipoD293 DNA transfection reagent for transfection of multiple mammalian cell lines (such as Hela and PC3) with luciferase reporters, you will get much higher luciferase readings than other transfected reagents used on our lab for luciferase reading, but observe little cell death in transfected cells In our condition! 1) Use the same type of culture medium (but unsupplemented) for diluting lipoD293 reagent and luciferase reporter DNA (for example, if the cells are cultured in RPMI1640-based medium, then use unsupplemented RPMI1640 as dilution medium); 3) No need to change medium either before or after transfection; 4) Add diluted lipoD293 reagent to diluted DNA and then incubate the mixture at room temperature for 15~20 minutes; 5) Make lysates for luciferase assay 16~48hrs post transfection siRNA transfection in vitro Optimization of GenMute TM siRNA in vitro transfection GenMute TM siRNA Transfection Reagent (Cat # SL100568) is one of the market's most potent siRNA delivery vehicle. Optimal siRNA concentrations ranged from 1.0 nM to 10 nM, and excessive siRNA may result in a "flood effect" with poor silencing. Our laboratory has been used successfully GenMute TM Transfection Reagent knockout expression of endogenous growth factors. 24-well plates, for example, the following steps will be how to optimize GenMute TM Transfection Reagent do a guide: As for other specifications utensils cultured cells, please refer to the experiments show GenMute TM. 1. Change the medium 30 to 60 minutes before transfection and add 0.5 ml of complete medium (containing serum and antibiotics) to each well. 2. 2.0 pmol siRNA (5.0 μM x 4.0 μl) was added to a sterile tube containing 200 μl of GenMute Transfection Buffer (Cat# SL100572) for dilution and allowed to stand at room temperature for 5 minutes. 3. Add 2.0 μl of GenMute transfection reagent, mix and place at room temperature for 15 minutes to form a transfection complex. Please note: The transfection time of the transfection complex should not exceed 25 minutes at room temperature. 4, 50μl, 25μl, 12.5μl transfection complex were added to the cells (re-well), the final concentration of siRNA reached 10nM, 5.0nM, 1.25nM. 5. After transfection for 24 to 48 hours, the transfection efficiency at 4 different siRNA concentrations was tested and the optimal transfection conditions were selected. Optimization of GenMuteTM reagent for siRNA silencing. GenMuteTM siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations range from 1.0 nM to 10 nM. Excessive siRNA may lead to "flooding effect" with sub-optimal silencing. Our lab has been using GenMuteTM reagent to knock down endogenously expressed growth factors with very good luck. The following procedures will guide to optimize GenMuteTM reagent for best silencing in 24-well plate. For other cell culture formats, please refer to the protocol Of GenMuteTM reagent. How to optimize GenMute TM transfection reagent to improve siRNA/DNA co-transfection efficiency GenMute TM siRNA Transfection Reagent (Cat # SL100568) is one of the market's most potent siRNA delivery vehicle. When siRNA/DNA is co-transfected, the optimal concentration range of siRNA is 0.5ηM to 10ηM. Excessive siRNA may result in a "flood effect" with poor silencing effect. Do not use more than 20ηM siRNA. In 24-well plate as an example, how to optimize the steps will GenMute TM Transfection Reagent make a guide; The cultured cells other vessel specifications, please refer to the experiment described GenMute TM. 1. Change the medium 30-60 minutes before transfection and add 0.5 ml of complete medium (containing serum and antibiotics) to each well. 2. Add 0.5 μg of DNA to each of 4 sterile tubes containing 50 μl of GenMute Transfection Buffer (Cat# SL100572) for dilution, and then serially dilute the siRNAs in 4 tubes respectively····· 5.0 pmol of siRNA was added to the first tube, 2.5 pmol of siRNA was added to the second tube, 1.25 pmol of siRNA was added to the third tube, and 1.25 pmol of siRNA was added to the fourth tube. Allow to stand at room temperature for 5 minutes. 3, taken 3.0μl GenMute TM Transfection Reagent were added to the diluted siRNA / DNA tubes, mixed and allowed to stand for 15 minutes to form a transfection complex at room temperature. Please note: The transfection time of the transfection complex should not exceed 25 minutes at room temperature. 4. The transfection complexes prepared above were separately added to four consecutive wells in a 24-well plate. 5. After 24-48 hours of transfection, the silencing effect in 4 wells was tested and the optimal transfection conditions were selected. Optimization of GenMuteTM reagent for siRNA/DNA co-transfection. GenMuteTM siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations for siRNA/DNA co-transfection range from 0.5 nM to 10 nM. Excessive siRNA may lead to "flooding effect" "The following procedures will guide to optimize GenMuteTM reagent for best siRNA/DNA co-transfection in 24-well plate. For other cell culture formats, please refer to the Protocol of GenMuteTM reagent. 2. Dilute 0.5 μg DNA to each sterile tube of total 4 tubes with 50 μl of GenMute transfection buffer (Cat # SL100572) followed by addition series diluted siRNA to each well of the total 4 tubes-------add 5.0 pmol siRNA to 1st tube, 2.5 pmols to 2nd tube, 1.25 pmols to 3rd tube and 1.25 pmols to 4th tube. Let's sit at RT for 5 minutes. 3. Add 3.0 μl GenMuteTM reagent to diluted siRNA/DNA of each tube, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT. 4. For 4 consecutive wells of 24-well plate, add the transfection complexes which are prepared with same concentration of DNA (0.5 μg per well) and different 4 concentrations of siRNA ranging from final 10 nM to 1.25 nM. 5. Check silencing effect in the 4 wells 24~48 hours post transfection and choose the best transfection conditions. How to effectively reduce the toxicity of PepMute TM , GenMute TM and PepMute TM Plus siRNA transfection reagents PepMute TM, GenMute TM and PepMute TM Plus transfection reagent toxicity was found only occasionally in DMEM basal cell culture medium, the use of other media (containing 10% FBS and antibiotics) growing the cells, the transfected substantially reduced toxicity. For example, if you find a large number of cell deaths, you may use DMEM basal medium with 10% FBS and antibiotics. To eliminate toxicity, you can choose DMEM/F12 or RPMI 1640 instead of DMEM as the base medium. As for why, so far, we are not known, but it has effectively reduced toxicity. Try it! How to eliminate toxicity of PepMuteTM, GenMuteTM and PepMuteTM Plus siRNA Reagents PepMuteTM, GenMuteTM and PepMuteTM Plus siRNA transfection reagents are found to be sometimes toxic ONLY with DMEM base cell culture medium. Using other cell culture mediums (supplemented with 10% FBS and antibiotics) to grow your cells will basically eliminate the transfection toxicity For instance, if you find toxicity and high cell death, you are probably using DMEM base medium supplemented with 10% FBS and antibiotics to grow your cells. To remove the transfection toxicity, you can change DMEM to DMEM/F12 or RPMI 1640 as Base medium. So far we have not figured out why, but it does minimize the toxicity. Try it out! 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We prepared pEGFP-N3 plasmid with Endofree Maxi Plasmid Kit (Qiagen), PerfectPrep Endofree Plasmid Maxi Kit (5 PRIME, Inc.) and NucleoBond DNA Maxi Kit (MACHEREY-NAGEL GmbH & Co. KG). The DNA quality was determined by measuring Absorbance at 230, 260, 280 and 320 nm by spectrometry.
The purity of DNA prepared by the three methods is as follows:
MACHEREY-NAGEL > 5 Prime > Qiagen.
Now we like to share some technical points which I think are important for maximum efficiency and minimal toxicity and often ignored:
Follow the following technical tips to choose the proper diluent for GenJetTM, PolyJetTM and LipoD293TM DNA transfection reagents:
Use unsupplemented RPMI1640 / DMEM instead of Opti-MEM medium to dilute both lipoD293 and DNA (luciferase reporters), you will get further higher luciferase readings (one-fold increase) and save more money !
Several key points in the protocol:
1). Change medium and add 0.5 ml of complete medium each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.
2). Dilute 20 pmol siRNA (5.0 μM x 4.0 μl) into 200 μl of GenMute transfection buffer (Cat # SL100572) in a sterile tube and let's sit at RT for 5 minutes.
3). Add 2.0 μl GenMute reagent, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.
4). Add 50 μl transfection complex to your cells directly in duplicate (final 10 nM siRNA), 25 μl complex to another 2 wells (final 5.0 nM siRNA), 12.5 μl to 3rd 2 wells (final 2.5 nM siRNA) and 6.25 μl To 4th 2 wells (final 1.25 nM siRNA).
5). Check silencing effect in the 4 different siRNA concentrations 24~48 hours post transfection and choose the best transfection conditions.
1. Change medium and add 0.5 ml of complete medium to each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.